Daniela S Uroić

Grégory Baudouin

Laura A Ferguson

Hilary M Docherty

Ludovic Vallier

Kevin Docherty
email: k.docherty@abdn.ac.uk

A factor(s) secreted from MIN-6 β-cells stimulates differentiation of definitive endoderm enriched embryonic stem cells towards a pancreatic lineage +

Keywords: diabetes mellitus, stem cells, pancreatic differentiation, insulin gene, islets of Langerhans, Pdx1

A c c e p t e d M a n u s c r i p t 2

Abstract

In the mouse the developing pancreas is controlled by contact with, and signaling molecules secreted from, surrounding cells. These factors are best studied using explant cultures of embryonic tissue. The present study was undertaken to determine whether embryonic stem (ES) cells could be used as an alternative model in vitro system to investigate the role of cell-cell interactions in the developing pancreas.

Transwell culture experiments showed that MIN-6 β-cells secreted a factor or factors that promoted differentiation of ES cell derived definitive endoderm enriched cells towards a pancreatic fate. Further studies using MIN-6 condition medium showed that the factor(s) involved was restricted to MIN-6 cells, could be concentrated with ammonium sulphate, and was sensitive to heat treatment, suggesting that it was a protein or peptide. Further analyses showed that insulin or proinsulin failed to mimic the effects of the conditioned media. Collectively, these results suggest that β-cells secrete a factor(s) capable of controlling their own differentiation and maturation. The culture system described here presents unique advantages in the identification and characterisation of these factors.

Introduction

During embryogenesis in the mouse the pancreas first appears as evaginations on the dorsal and ventral surfaces of the primitive foregut at around embryonic day 9.5 (E9.5) [START_REF] Gittes | Developmental biology of the pancreas: a comprehensive review[END_REF]. This specification is associated with expression of the transcription factors Pdx1 and Ptf1a, which serve to divert cells away from duodenal, hepatic and duct fates [START_REF] Burlison | Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells[END_REF]. Once formed the stalk of the pancreatic buds elongates with branching morphogenesis occurring within the apical regions. Multipotent progenitors that give rise to all cell types in the pancreas are present at this branching stage, localised predominantly within the tip regions [START_REF] Zhou | A multipotent progenitor domain guides pancreatic organogenesis[END_REF]. At around E12 to E13 the gut rotates, the two buds come into contact with each other and fuse. During this period, termed the secondary transition [START_REF] Pictet | An ultrastructural analysis of the developing embryonic pancreas[END_REF], the multipotent progenitor cells begin to differentiate into the exocrine (acinar and duct) and endocrine cells of the pancreas.

The establishment and expression of the various cell lineages is controlled by the sequential expression of transcription factors [START_REF] Servitja | Transcriptional networks controlling pancreatic development and beta cell function[END_REF] in response to signalling molecules emanating from cells within the expanding pancreas and surrounding tissues. There are two possible types of signal, instructive and permissive. In a study that involved grafting blocks from various regions along the anterior-posterior axis from quail embryos into host chick embryos, it was demonstrated that lateral plate endoderm produced instructive signals that could induce pancreatic differentiation in naive endoderm [START_REF] Kumar | Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate[END_REF]. On the other hand notochord, endothelium and mesenchyme produce permissive signals [START_REF] Scharfmann | Beta-cell development: the role of intercellular signals[END_REF], i.e. signals that do not establish the pancreatic domain but act upon endoderm that has already been pre-patterned towards pancreas.

Experimental approaches aimed at characterising the signalling molecules that control development of the pancreas at present involve explant cultures of embryonic A c c e p t e d M a n u s c r i p t 4 tissues [START_REF] Gittes | Developmental biology of the pancreas: a comprehensive review[END_REF]. Thus growth factors or conditioned media would be added directly to the embryonic pancreatic tissue, or the tissue would be co-cultured with other cell types.

An alternative approach might be to use embryonic stem (ES) cells. A number of robust protocols have been developed whereby mouse and human ES cells can be induced to differentiate into β-like cells by mimicking the events that occur in the developing pancreas of the mouse embryo [START_REF] Docherty | Embryonic stem cell therapy for diabetes mellitus[END_REF]. The cells are first induced to form definitive endoderm (the germ layer which gives rise to the pancreas), and from there sequentially to pancreatic progenitors, endocrine cells, and β-cells. Although much progress has been made in generating insulin-secreting cells from ES cells, it has not been possible to generate a fully functional β-cell using currently available differentiation protocols [START_REF] Baetge | Production of beta-cells from human embryonic stem cells[END_REF]. However , if pancreatic progenitors generated from ES cells are grafted into mice, then over a prolonged period of time these cells mature into cells that exhibit many of the functional characteristics of β-cells [START_REF] Kroon | Pancreatic endoderm derived from human embryonic stem cells generates glucoseresponsive insulin-secreting cells in vivo[END_REF] demonstrating that fully functional pancreatic cells can be generated from pluripotent stem cells.

Here we describe how mouse ES cells that have been induced to form an enriched population of definitive endoderm can be used to identify and characterise factors secreted from MIN-6 β-cells that affect their further differentiation towards a pancreatic lineage. We anticipate that this in vitro system will have widespread applications in studies on differentiation factors secreted from other cell types, including those present in blood vessels and mesenchyme.

A c c e p t e d M a n u s c r i p t 5

Methods

Cell culture

The mouse embryonic stem cell line CGR8 [START_REF] Nichols | Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity[END_REF] was maintained in a pluripotent state in maintenance medium supplemented with leukaemia inhibitory factor (LIF) in tissue culture flasks coated with 0.1 % (w/vol) gelatin (Sigma-Aldrich). Maintenance medium consisted of knockout DMEM (Invitrogen) supplemented with 15 % (vol/vol) knockout serum replacement (Invitrogen), 0.1 mM MEM non essential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), and 140 µM 2mercaptoethanol (Sigma-Aldrich). MIN-6, a mouse β-cell line [START_REF] Miyazaki | Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms[END_REF]

Differentiation of CGR8 cells towards definitive endoderm

CGR8 cells were cultured as embryoid bodies (EBs) for 6 days in chemically defined medium (CDM) [START_REF] Wiles | Embryonic stem cell development in a chemically defined medium[END_REF]. Essentially this involved seeding CGR8 cells at a density of 1.3 x 10 6 cells per 6 cm bacterial grade Petri dish (Greiner Bio-one) and gently mixing them on a horizontal shaker for 2 days at 37˚C and 5 % CO 2 . The medium was then supplemented with activin A and/or BMP4 (R&D Systems) as indicated in the results section. EBs per well in growth factor reduced Matrigel™ (BD Biosciences) coated 6-well plates. The resultant outgrowths were cultured in CGR8 maintenance medium. In some experiments the outgrowths were co-cultured with MIN-6 cells using 0.4µm membrane transwells (Greiner Bio-One) in CGR8 maintenance medium.

Preparation of conditioned media

Conditioned media were prepared by incubating a 20-30% confluent culture of MIN-6, HEK-293, PANC-1 cells, and N2A cells in CGR8 maintenance medium.

Conditioned media were collected after 24 (CM24) or 48 (CM48) hours, filtered through a 0.22 µm filter, and stored at -80˚C until used.

Preparation of RNA and quantitative PCR

Preparation of RNA and quantitative PCR (qPCR) were performed as previously described [START_REF] Bernardo | Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells[END_REF]. Inventoried Taqman © gene expression assays (Applied Biosystems) were used for qPCR. Relative gene expression for each sample was determined using the Pfaffl formula [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. Randomisation tests to estimate p-values were done with REST© software [START_REF] Pfaffl | Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR[END_REF].

Ammonium sulphate precipitation

To generate a 70% fraction, ammonium sulphate powder was added slowly with stirring to conditioned medium at 4˚C. This was left without stirring for 15 -30 min A c c e p t e d M a n u s c r i p t 7 to allow all the salt to dissolve. The solution was then centrifuged at 5000 x g for 10 min. The supernatant was decanted, while the pellet was resuspended in 50 mM Trisacetate buffer, pH 7.0. If a second precipitation step was undertaken, ammonium sulphate powder was slowly added to the supernatant up to the second precipitation concentration. The solution was centrifuged as before, and the pellet dissolved in 50 mM Tris acetate buffer. Both samples were dialysed four times against at least 100 volumes of 50 mM Tris acetate buffer.

Protein assay

Protein concentrations were determined using the DC protein assay (Bio Rad) according to the manufacturer's instructions.

Immunocytochemistry

Results

ES cells can be induced to form endoderm by treatment with activin A as a surrogate

for nodal signalling [START_REF] Kubo | Development of definitive endoderm from embryonic stem cells in culture[END_REF][START_REF] D'amour | Efficient differentiation of human embryonic stem cells to definitive endoderm[END_REF][START_REF] Hansson | A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells[END_REF]. Thus, when ES cells (CGR8) were cultured as embryoid bodies (EBs), activin A at 30 ng/ml and 100 ng/ml increased the levels of the definitive endoderm markers Sox17, Foxa2 and CXCR4, while decreasing the expression of the mesoderm marker Bry (Fig. 1a). Although BMP-4 is a powerful inducer of mesoderm [START_REF] Johansson | Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development[END_REF] we found in our previous study [START_REF] Bernardo | Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells[END_REF] and in the present study that prior treatment with BMP-4 before activin A increased the levels of the definitive endoderm markers, while reducing the levels of the extraembryonic endoderm markers Sox7 and Afp, which remained elevated with activin A alone.

Treatment with BMP-4 for 3 days followed by activin A for 3 days (B,A) also decreased the levels of the mesoderm marker Bry and the ectoderm marker Zic1, but like activin A alone had little effect on the primitive streak markers Mixl1 and Gsc.

We were also able to show by immunocytochemistry that the percentage of Sox17 and CXCR positive cells following B,A treatment was similar to that following treatment with activin A alone [START_REF] Bernardo | Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells[END_REF]. For these reasons all subsequent experiments were performed on a definitive endoderm enriched population generated using BMP-4 and activin A, because these conditions generated the most consistent decrease in non definitive endoderm markers.

To determine whether MIN-6 β-cells secreted factors that could affect the differentiation of ES cells towards a pancreatic lineage, the definitive endoderm enriched EBs were cultured as outgrowths in transwell culture dishes in the presence or absence of MIN6-cells. Following differentiation in the transwell dishes for 7 days (DD7), most of the cells within, and especially towards the periphery of the EB, stained positively for the transcription factor Nkx6.1 (Fig. 2A) with no detectable A c c e p t e d M a n u s c r i p t 9 staining for Pdx1 (Fig. 2B). At day 14 there were scattered clusters of cells within the outgrowth that co-stained for Nkx6.1 and Pdx1 and for insulin and Pdx1 (Fig 2, E-L).

These cell clusters increased in number from day 7 to day 14, and although they could rarely be detected in cultures in the absence of MIN-6 cell, their presence was increased significantly by the MIN-6 cells (Fig. 3). These results suggested that the MIN-6 cells were secreting a factor(s) that was stimulating the production of clusters of pancreatic endocrine cells in the DE-enriched outgrowths.

In order to characterise the pancreatic differentiation factor(s) secreted from MIN-6 cells the DE-enriched outgrowths were cultured in KO DMEM (control media) or the same media that had been exposed to MIN-6 cells for 24 or 48 hours.

The results showed that the conditioned media stimulated the expression of the early pancreatic transcription factors Hnf1β, Pdx1 and Ptf1a (Fig. 4), suggesting that CM from MIN-6 cells could stimulate differentiation of endoderm cells into pancreatic progenitors. We also observed that CM from MIN-6 cells was able to increase the expression of  cells markers (insulin 1, insulin 2, islet amyloid polypeptide (IAPP), glucose transporter 2 (GLUT2)), and alpha cell (glucagon), delta cell (somatostatin) and, acinar cell (amylase) markers (Fig. 4), which is in keeping with the stimulated appearance of insulin positive cells in the transwell experiments. To confirm this stimulatory effect EBs were grown for up to 19 days (Fig. 5) in the presence of CM from MIN-6 and Q-PCR showed a strong and robust increase in Pdx1, insulin 1, insulin 2 and glucagon expression. In addition, the effect was dose-dependent (Fig. 6).

We were unable to detect insulin or Pdx1 by immunocytochemistry in CM treated cultures, which was in keeping with the low Ct values. This probably relates to differences between the CM and transwell experiments: in the transwell experiments the DE-enriched cells were treated with higher amounts of newly synthesised factor(s)

A c c e p t e d M a n u s c r i p t 10 for longer periods of time. However, taken together, the CM results confirmed that βcells could produce a factor(s) capable of promoting pancreatic differentiation of ESderived definitive endoderm. Importantly, these results could not be explained by uptake of mRNA from the conditioned media. Indeed, we could not detect mRNA for insulin 1, insulin 2 and Pdx1 in the conditioned media, suggesting that the filtration step had efficiently removed dead cells and any debris with which nucleic acids might be associated.

The pancreatic differentiation factor(s) appeared to be β-cell specific in that it was absent from HEK-293, Panc-1 and N2A cells (Fig. 7). Interestingly, conditioned media from HEK-293 had a marked stimulatory effect on the insulin 2 gene but not the insulin 1 gene. This may be explained by the fact that, whereas insulin 1 is expressed predominantly in β cells during embryogenesis, insulin 2 is expressed in cells of the nervous system, liver and yolk sac [START_REF] Deltour | Differential expression of the two nonallelic proinsulin genes in the developing mouse embryo[END_REF][START_REF] Devaskar | Insulin gene expression and insulin synthesis in mammalian neuronal cells[END_REF].

In order to characterise the MIN-6 cell factor(s) the conditioned media was subjected to ammonium sulphate precipitation. Expression of both insulin 1 and insulin 2 was stimulated by the 70% ammonium sulphate fraction (Fig. 8). There was some activity present in the 100% ammonium sulphate fraction and this activity was most pronounced upon the insulin 2 gene. Notably, an 85% ammonium sulphate fraction stimulated the expression of insulin 1 and insulin 2 to the same extent as the conditioned medium (Fig. 8). These results suggested that the factor(s) was a protein or small peptide that could be inactivated by heat treatment at 95 o C for 30 minutes (Fig. 8).

Of all the proteins secreted by a MIN-6 cell, insulin is by far and away the most abundant. We therefore tested whether insulin at physiological concentrations, 2.4 and 5.8 ng/ml, or in excess, i.e. 7 μg/ml, could mimic the effects of the MIN-6

A c c e p t e d M a n u s c r i p t 11 conditioned media. The results showed a complete lack of effect of exogenous insulin on the expression of insulin 1 or insulin 2 at either low or high concentrations (Fig. 9).

Although the MIN-6 cells can efficiently process proinsulin to insulin, the small amount of proinsulin in the media could be responsible for the differentiation activity.

We therefore tested the effect of recombinant human proinsulin. The results showed that, like insulin, the exogenous proinsulin had no effect on the expression of insulin 1, or insulin 2 (Fig. 10). Together, these results demonstrate that neither insulin nor proinsulin is the secreted factors that are capable of increasing pancreatic differentiation of ES-derived definitive endoderm

Discussion

The major finding of the study was that a factor or factor(s) secreted by in MIN-6 βcells could enhance differentiation of definitive endoderm enriched cells towards a pancreatic lineage, suggesting that β-cells control part of their own development and maturation. This confirms and extends previous findings that pancreatic extracts [START_REF] Choi | In vitro transdifferentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract[END_REF][START_REF] Gabr | Transplantation of insulin-producing clusters derived from adult bone marrow stem cells to treat diabetes in rats[END_REF] and adult islets cells [START_REF] Chen | Promotion of beta-cell differentiation in pancreatic precursor cells by adult islet cells[END_REF] may provide signals to precursor cells that affect differentiation to endocrine pancreatic lineages.

The presence of the MIN-6 cells or CM stimulated expression of both early and late pancreatic markers, which is in keeping with evidence that β-like (i.e. insulin positive) cells are present in two waves during embryogenesis in the mouse. The first wave begins at about E9.5 with the appearance of insulin + , glucagon + , and cells that stain double positive for both insulin and glucagon [START_REF] Pictet | An ultrastructural analysis of the developing embryonic pancreas[END_REF]. These early insulin + cells lack many of the markers of mature β cells [START_REF] Pang | Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing rat pancreas[END_REF], and it is unlikely that these cells contribute to mature β-cells and their fate is unclear [27]. Our results on the effect of The second wave of β-cell formation occurs during the secondary transition after E13. A recent study has shown that β-cells present during this stage of development may interact with other endocrine cells to establish the correct proportion of endocrine cells [START_REF] Gannon | Pdx-1 Function is Specifically Required in Embryonic Beta Cells to Generate Appropriate Numbers of Endocrine Cell Types and Maintain Glucose Homeostasis[END_REF]. Thus a conditional knockout of Pdx1 at late gestation prior to islet formation led to a dramatic reduction in insulin + cells and an increase in glucagon + and somatostatin + cells in the neonatal islets. These paracrine effects of the β-cell on the adjacent endocrine cells are not particularly rapid, since in another study that involved toxin-mediated ablation of β-cells no effect on the development of glucagon + and somatostatin + cells was observed at E16.5 [START_REF] Herrera | Ablation of islet endocrine cells by targeted expression of hormone-promoter-driven toxigenes 361[END_REF]. Taken together these results clearly demonstrate the existence of a factor(s) produced by βcells themselves that influence their differentiation and maturation.

Importantly, the in vitro culture system described here presents unique advantages toward identifying these factors. It allows one to undertake large scale experiments impossible to perform in vivo, and also to screen efficiently for the effects of potential candidates. As an example, this system already allowed us to demonstrate that the factor(s) was absent from other cells lines that were tested and was likely to be a protein or small peptide based on ammonium sulphate precipitation and heat stability. In addition, the factor was not insulin since exogenous insulin had no effect on the differentiation of the definitive endoderm enriched cells. This is not unexpected since high concentrations of insulin are normally present in media used to culture and differentiate ES cells, and there is no evidence that insulin can enhance formation of pancreatic lineages. The lack of effect of proinsulin further suggests that the factor is unlikely to be a product of the insulin gene, which would be in keeping A c c e p t e d M a n u s c r i p t 13 with previous observations that a double mutation for insulin 1 and insulin 2 had no major developmental defects, although the islets were slightly larger [START_REF] Duvillie | Phenotypic alterations in insulin-deficient mutant mice[END_REF][START_REF] Duvillie | Increased islet cell proliferation, decreased apoptosis, and greater vascularization leading to beta-cell hyperplasia in mutant mice lacking insulin[END_REF].

Interestingly, it was previously shown that conditioned media from pancreatic fibroblasts were able to induce β-cell proliferation and formation of islet-like structures. These effects were specifically blocked using an anti-HGF antibody [START_REF] Otonkoski | Hepatocyte growth factor/scatter factor has insulinotropic activity in human fetal pancreatic cells[END_REF].

Therefore, it would be interesting to test if HGF can mimic the effect of MIN-6 conditioned media and if this effect can be blocked by anti-HGF antibodies.

In summary, our results suggest that the developing β-cell may secrete a factor(s) that affects the development of the pancreas. The culture system described here will facilitate the purification and characterisation of this factor(s) and allow us to determine whether it is present and capable of functioning during the first and/or second waves of β-cell expression. Such a factor(s) would have important applications in generating a replenishable supply of β-cells from ES or iPS cells. The ES differentiation system may also be of use in characterising pancreatic differentiation factors from endothelial and mesenchymal cell populations that are known to secrete factors that control cell fate during pancreatogenesis. ES cells were cultured as EBs in CDM for 6 days with 30 ng/ml activin A (30 A), 100 ng/ml activin A (100A), 1 ng/ml BMP-4 for 3 days followed by 100 ng/ml activin A for 3 days (B,A), and 10 ng/ml BMP-4 and 100 ng/ml activin for 3 days followed by 100 ng/ml activin A for 3 days (AB,A). The untreated ES cells (ESC) are also shown.

  was maintained in high glucose DMEM (Invitrogen) supplemented with 15 % foetal calf serum (FCS) (Invitrogen) and 70 µM 2-mercaptoethanol (Sigma-Aldrich). PANC-1, a human pancreatic carcinoma, epithelial-like cell line (ATCC® Number: CRL-1469™) was maintained in high glucose DMEM supplemented with 10 % FCS and 4 mM Lglutamine (Invitrogen). HEK-293, a human foetal kidney cell line (ATCC ® Number: CRL-1573™) and N2A, a rat neuronal cell line (ATCC ® Number: CCL-131™), were maintained in low glucose DMEM supplemented with 10% FCS,

A c c e p t e d M a n u s c r i p t 6

 6 Differentiation of definitive endoderm enriched cultures towards pancreatic endodermDefinitive endoderm enriched embryoid bodies (EB)s were seeded at a density of 30

  Cells were fixed with 4% paraformaldehyde solution (PFA), and permeabilised with 0.2% Triton. The following antibodies were used: guinea pig anti-insulin (Abcam) at 1/125, goat anti-Nkx6.1 (R&D) at 1/100, and rabbit anti-Pdx1 (kindly provided by Dr C. Wright, Vanderbilt University) at 1/400.

A c c e p t e d M a n u s c r i p t 12 MIN- 6

 126 media on early markers of pancreatic differentiation suggest that these "vestigial" β-cells may function to regulate the early stages of pancreatic formation.

Figure 1 .

 1 Figure 1. Effect of activin A and BMP-4 on the differentiation of mouse ES cells.

  The cells were harvested, RNA prepared and the indicated mRNA levels were measured by qPCR. The data are expressed as fold change in gene expression relative to GAPDH and normalised to an untreated control (N/A). The results represent mean ± SD where n=3 (* p<0.05, ** p<0.01, *** p<0.001 relative to the untreated control).

Figure 2 .

 2 Figure 2. Min-6 cells secrete a factor(s) that stimulates differentiation towards a pancreatic lineage. DE-enriched outgrowths were co-cultured with MIN-6 cells in 0.4 μM transwell dishes. After 7 or 14 days of differentiation (DD7 or DD14) the cells were stained with the indicated antibodies. The staining in panels A-D are from DD7 cultures and show an attached EB with some outgrowth. Panels E-L show staining within the outgrowth which now covers a substantial portion of the dish. The data shown here are from a single experiment that was repeated on three separate occasions with similar results.

Figure 3 .

 3 Figure 3. The presence of MIN-6 cells stimulates the formation of cell clusters. DE-enriched outgrowths were co-cultured with MIN-6 cells in 0.4 μM transwell dishes. After 7 or 14 days of differentiation (DD7 or DD14) the cells were stained with the indicated antibodies and number of antibody positive cell clusters counted.

A c c e p t e d M a n u s c r i p t 18 The

 18 results represent the mean ± SEM of three separate wells of a 6-well dish. The experiment was repeated 3 times with a similar result. * p<0.05 relative to cells incubated without MIN6 cells.

Figure 4 .

 4 Figure 4. MIN-6 conditioned media induces differentiation of definitive endoderm enriched cells towards pancreas. Definitive endoderm enriched cells were cultured for 10 days as a monolayer in DMEM (N/A) or condition medium from 24h (CM24) or 48h (CM48) cultures of MIN-6 cells. The cells were harvested, RNA prepared and the indicated mRNA levels were measured by qPCR. The data are expressed as fold change in gene expression relative to GAPDH and normalised to an untreated control (N/A). The results represent mean ± SD where n=3 (* p<0.05, ** p<0.01, *** p<0.001 relative to the untreated control).

Figure 5 .

 5 Figure 5. Prolonged exposure to MIN-6 conditioned medium enhances the stimulatory effect on pancreatic markers. Definitive endoderm enriched cells were cultured as a monolayer for 10, 15 or 19 days in DMEM (N/A) or conditioned medium from 48-hour cultures of MIN-6 cells (CM). The cells were harvested, RNA prepared and the indicated mRNA levels were measured by qPCR. The data are expressed as fold change in gene expression relative to GAPDH and normalised to an untreated control (N/A). The results represent mean ± SD where n=3 (* p<0.05, ** p<0.01, *** p<0.001 relative to the untreated control).

A c c e p t e d M a n u s c r i p t 19 Figure 6 .

 196 Figure 6. The effect of MIN-6 conditioned media is dose dependent. Definitive endoderm enriched cells were cultured as a monolayer for 10 days in DMEM (N/A) or neat (1x) or four times concentrated conditioned medium (4x) from 48-hour cultures of MIN-6 cells. The cells were harvested, RNA prepared and insulin 1 (ins1) and insulin 2 (ins2) mRNA levels were measured by qPCR. The data are expressed as fold change in gene expression relative to GAPDH and normalised to an untreated control (N/A). The results represent mean ± SD where n=3 (* p<0.05, ** p<0.01, *** p<0.001 relative to the untreated control).

Figure 7 .

 7 Figure 7. The pancreatic differentiation factor(s) is restricted to MIN-6 cells. Definitive endoderm enriched cells were cultured as a monolayer for 10 days in DMEM (N/A) or conditioned medium from 48-hour cultures of MIN-6, Panc-1, HEK-293 and N2A cells. The cells were harvested, RNA prepared and the indicated mRNA levels were measured by qPCR. The data are expressed as fold change in gene expression relative to GAPDH and normalised to an untreated control (N/A). The results represent mean ± SD where n=3 (* p<0.05, ** p<0.01, *** p<0.001 relative to the untreated control).

Figure 8 .

 8 Figure 8. The pancreatic differentiation factor(s) can be concentrated using ammonium sulphate.Definitive endoderm enriched cells were cultured as a monolayer for 10 days in DMEM (N/A) or conditioned medium from 48-hour cultures of MIN-6 cells, or with a 70% (F70), 85% (F85) or 100% (F100) ammonium sulphate fraction from the condition medium. F70 + F100 contains a combination of the F70 and F100 fractions.The effect of the heat-treated F70 fraction is also shown. The cells were harvested,

Figure 9 .

 9 Figure 9. The pancreatic differentiation factor(s) is not insulin. Definitive endoderm enriched cells were cultured as a monolayer for 10 days in DMEM (N/A) or conditioned medium from 48-hour cultures of MIN-6 cells (CM), or supplemented with exogenous insulin at the indicated concentrations. The cells were harvested, RNA prepared and the indicated mRNA levels were measured by qPCR. The data are expressed as fold change in gene expression relative to GAPDH and normalised to an untreated control (N/A). The results represent mean ± SD where n=3 (* p<0.05, ** p<0.01, *** p<0.001 relative to the untreated control).

Figure 10 .

 10 Figure 10. The pancreatic differentiation factor(s) is not proinsulin. Definitive endoderm enriched cells were cultured as a monolayer for 10 days in DMEM (N/A) or conditioned medium from 48-hour cultures of MIN-6 cells (CM), or supplemented with exogenous proinsulin at the indicated concentrations. The cells were harvested, RNA prepared and the indicated mRNA levels were measured by qPCR. The data are expressed as fold change in gene expression relative to GAPDH and normalised to an untreated control (N/A). The results represent mean ± SD where n=3 (* p<0.05, ** p<0.01, *** p<0.001 relative to the untreated control).

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Acknowledgements

This project was funded by the JDRF. LV is supported by a MRC senior non-clinical fellowship. DSU was funded by a research studentship from Diabetes UK and GB by Adam's Scholarship.


Mervi E Hyvönen

Pauliina Saurus

Anita Wasik

Eija Heikkilä

Marika Havana

Ras Trokovic

Moin Saleem

Harry Holthöfer

Sanna Lehtonen
email: sanna.h.lehtonen@helsinki.fi

Lipid phosphatase SHIP2 downregulates insulin signalling in podocytes

Keywords: diabetes, diabetic nephropathy, insulin resistance, podocyte, proteinuria

Podocyte injury plays an important role in the development of diabetic nephropathy.

Podocytes are insulin responsive and can develop insulin resistance, but the mechanisms are unknown. To study the role of CD2-associated protein (CD2AP) in podocyte injury, we performed a yeast two-hybrid screening on a glomerular library, and found that CD2AP bound to SH2-domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2), a negative regulator of insulin signalling. SHIP2 interacts with CD2AP in glomeruli and is expressed in podocytes, where it translocates to plasma membrane after insulin stimulation. Overexpression of SHIP2 in cultured podocytes reduces Akt activation in response to insulin, and promotes apoptosis. SHIP2 is upregulated in glomeruli of insulin resistant obese Zucker rats. These results indicate that SHIP2 downregulates insulin signalling in podocytes. The upregulation of SHIP2 in Zucker rat glomeruli prior to the age of onset of proteinuria suggests a possible role for SHIP2 in the development of podocyte injury.

A c c e p t e d M a n u s c r i p t

Introduction

Diabetic nephropathy is one of the leading causes of end stage renal disease and kidney transplantations, and is also associated with an increased risk for cardiovascular morbidity and mortality [START_REF] Borch-Johnsen | The natural history of insulin-dependent diabetes mellitus in Denmark: 1. Long-term survival with and without late diabetic complications[END_REF]. Diabetic nephropathy is characterized by early development of proteinuria indicating an injury in the glomerular filtration barrier, which consists of three layers: fenestrated endothelial cells, basement membrane, and glomerular epithelial cells or podocytes. Podocytes are highly specialized cells forming foot processes that surround the glomerular capillary walls and are interconnected with special cell-adhesion structures called slit diaphragms. One of the key components of the slit diaphragm is nephrin, a transmembrane protein of the immunoglobulin superfamily, mutated in the congenital nephrotic syndrome of the Finnish type [START_REF] Kestila | Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital nephrotic syndrome[END_REF]. Interestingly, nephrin has been shown to be downregulated in diabetic nephropathy [START_REF] Langham | Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition[END_REF].

It has become evident that podocyte injury plays an important role in the development of diabetic nephropathy like in many other proteinuric diseases [START_REF] Wolf | From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy[END_REF]. Podocyte number is reduced already after short duration of diabetes in renal biopsies from patients with type 1 diabetes [START_REF] Steffes | Glomerular cell number in normal subjects and in type 1 diabetic patients[END_REF] and podocyte loss is also described in type 2 diabetes where it correlates with disease progression [START_REF] Meyer | Podocyte number predicts long-term urinary albumin excretion in Pima Indians with Type II diabetes and microalbuminuria[END_REF]. Podocyte foot process effacement [START_REF] Steffes | Epithelia cell foot process width in intact and uninephrectomized diabetic and nondiabetic rats[END_REF][START_REF] Ellis | Observations of glomerular epithelial cell structure in patients with type I diabetes mellitus[END_REF] and detachment from the basement membrane [START_REF] Toyoda | Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy[END_REF] are seen in both human patients and animal models. In addition to detachment, podocyte loss can result from increased apoptosis [START_REF] Verzola | Apoptosis in the kidneys of patients with type II diabetic nephropathy[END_REF] which precedes the increase in urinary albumin excretion in mouse models of diabetes (Susztak et al 2006).

Insulin resistance is known to be associated with microalbuminuria in diabetic patients [START_REF] Yip | Insulin resistance in insulin-dependent diabetic patients with microalbuminuria[END_REF][START_REF] Parvanova | Insulin resistance and microalbuminuria: a crosssectional, case-control study of 158 patients with type 2 diabetes and different degrees of urinary albumin excretion[END_REF]. Further, in type 1 diabetic patients insulin resistance has shown to predict [START_REF] Orchard | Nephropathy in type 1 diabetes: a manifestation of insulin resistance and multiple genetic susceptibilities? Further evidence from the Pittsburgh Epidemiology of Diabetes Complication Study[END_REF] and the metabolic syndrome to associate with (Thorn et al 2005) overt nephropathy, but the mechanisms have remained unsolved. Podocytes have shown to be insulin-responsive and to increase their glucose A c c e p t e d M a n u s c r i p t uptake after insulin stimulation [START_REF] Coward | The human glomerular podocyte is a novel target for insulin action[END_REF]. This suggests that insulin resistance can have a direct effect on podocyte function. Indeed, in a recent article Tejada et al.

showed that podocytes from diabetic db/db mice were unable to phosphorylate Akt in response to insulin (Tejada et al 2008).

CD2-associated protein (CD2AP) is a cytosolic adaptor protein with multiple proteinprotein interaction domains [START_REF] Dustin | A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts[END_REF]. It interacts with nephrin [START_REF] Palmen | Interaction of endogenous nephrin and CD2-associated protein in mouse epithelial M-1 cell line[END_REF], and as CD2AP associates with actin directly [START_REF] Lehtonen | CD2-associated protein directly interacts with the actin cytoskeleton[END_REF], it has been suggested to link nephrin to the actin cytoskeleton. Mice lacking CD2AP develop nephrotic syndrome, indicating that CD2AP is required to maintain the functional integrity of the glomerular slit diaphragm [START_REF] Shih | Congenital nephrotic syndrome in mice lacking CD2-associated protein[END_REF]. Heterozygous mice lacking one allele of CD2AP develop glomerular abnormalities at an advanced age and are more susceptible to glomerular damage than wild-type mice [START_REF] Kim | CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility[END_REF]. CD2AP mutations have also been reported in human patients with focal segmental glomerulosclerosis [START_REF] Kim | CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility[END_REF].

In order to study the role of CD2AP in podocyte injury, we performed a yeast-two-hybrid screening of a glomerular library. Interestingly, we found that CD2AP bound to SH2domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2), a negative regulator of insulin signalling and a molecule associated with the metabolic syndrome [START_REF] Dyson | The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2[END_REF]. Lipid phosphatase SHIP2 downregulates PI3K mediated signalling, induced by insulin and other growth factors, by hydrolyzing PI(3,4,5,)P 3 to PI(3,4)P 2 [START_REF] Blero | The SH2 domain containing inositol 5-phosphatase SHIP2 controls phosphatidylinositol 3,4,5-trisphosphate levels in CHO-IR cells stimulated by insulin[END_REF], Pesesse et al 2001). SHIP2 overexpression in mice has a negative effect on glucose tolerance [START_REF] Kagawa | Impact of transgenic overexpression of SH2containing inositol 5'-phosphatase 2 on glucose metabolism and insulin signaling in mice[END_REF][START_REF] Fukui | Impact of the liver-specific expression of SHIP2 (SH2-containing A c c e p t e d M a n u s c r i p t inositol 5'-phosphatase 2) on insulin signaling and glucose metabolism in mice[END_REF] and SHIP2 knockout mice appear resistant to high fat diet-induced obesity and insulin resistance [START_REF] Sleeman | Absence of the lipid phosphatase SHIP2 confers resistance to dietary obesity[END_REF], and in addition, inhibition of SHIP2 in the liver has been shown to improve glucose tolerance in diabetic mice [START_REF] Grempler | Normalization of prandial blood glucose and improvement of glucose tolerance by liver-specific inhibition of SH2 domain containing inositol phosphatase 2 (SHIP2) in diabetic KKAy mice: SHIP2 inhibition causes insulin-mimetic effects on glycogen metabolism, gluconeogenesis, and glycolysis[END_REF]. Taken together, SHIP2 is a downregulator of PI3K mediated insulin signalling, that is known to function in liver, muscle and adipose cells, but its role in the kidney has not been studied previously. We hypothesized that SHIP2 can mediate insulin resistance in podocytes, and investigated its expression and A c c e p t e d M a n u s c r i p t function in cultured podocytes and in diabetic rat and mouse kidneys.

Materials and methods

Yeast two-hybrid screening

Construction of the rat glomerular yeast two-hybrid library has been described previously (Takeda et al 2001). The cDNA encoding the N-terminal part of mouse CD2AP (aa 1-330) was amplified by PCR and subcloned into NcoI and XhoI sites in the LexA DNAbinding domain-containing displayBAIT vector (Display System Biotech, Vista, CA).

The displayBAIT-CD2AP and the displayREPORTER vector, containing green fluorescent protein gene as a reporter, were transformed into yeast strain EGY48 (Invitrogen, Carlsbad, CA). 50 µg of the rat glomerular yeast two-hybrid library in the displayTARGET vector was transformed into yeast pretransformed with displayBAIT-CD2AP and displayREPORTER vectors and plated on selective medium (-histidine,uracil, -leucine, -tryptophane). Colonies growing in the selective medium were exposed to UV light (360 nm) to select the ones expressing the green fluorescent protein reporter.

Plasmid DNA of the positive clones was electroporated into Escherichia coli KC8 (Clontech, Mountain View, CA) and the identity of the inserts was analyzed by sequencing and Blastn (nucleotide-nucleotide BLAST) search.

Animals

Male Sprague-Dawley rats and male FVB mice were used to study SHIP2 expression in normal rat and mouse. Male db/db mice and heterozygous db/+ controls in C57BLKS background (BKS.Cg-m+/+Lepr db /BomTac) were purchased from Taconic Europe (Ry, Denmark), and obese (fa/fa) and lean (fa/+) Zucker rats (Crl:ZUC-Leprfa) from Charles River Laboratories (Sulzfeld, Germany). Animal care followed the guidelines of NIH and Finnish laws. Blood glucose values were measured from tail vein samples using OneTouch Ultra glucometer (Lifescan, Milpitas, CA). The urinary albumin to creatinine ratio was determined from spot urine samples. Albumin was measured with mouse or rat albumin ELISA kit (CellTrend, Luckenwalde, Germany) and creatinine using CREA plus A c c e p t e d M a n u s c r i p t enzymatic assay (Roche, Basel, Switzerland) and Roche clinical chemistry analyzer. For histological analysis kidneys were fixed in 10% formalin, embedded in paraffin and analyzed by hematoxylin-eosin staining.

Cell culture

Immortalized human podocytes [START_REF] Saleem | A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression[END_REF] were maintained as previously described in RPMI medium, supplemented with 10% FBS, and insulin, transferrin and selenite (ITS), at + 33 C for proliferation and termo-switched to + 37 C for differentiation for 14 days. HIRc cells (rat fibroblasts overexpressing human insulin receptor) [START_REF] Mcclain | A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis[END_REF] were maintained in DMEM medium, supplemented with 10% FBS, ultraglutamine, penicillin and streptomycin. All cell culture reagents were from Sigma ( St. Louis, MO) except ultraglutamine from Lonza (Basel, Switzerland).

Immunoblotting

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and scraped from the dish into ice-cold 1% Nonidet P-40 (NP-40), 20 mM Hepes, pH 7.5, 150 mM NaCl with 1x Complete™, EDTA-free proteinase inhibitor cocktail (Roche), 50 mM NaF and 1 mM Na 3 VO 4 (lysis buffer) and rotated at 4°C for 15 min. Glomeruli and tubules were isolated from rat kidney cortices by graded sieving and lysed by rotation in lysis buffer at 4°C for 30 min. Detergent insoluble material was removed by centrifugation (10,000 x g for 10 min). For immunoblotting proteins were separated by SDS-PAGE and transferred to PVDF-FL membranes (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) diluted 1:1 with PBS, and incubated with goat anti-SHIP2 I-20 (Santa-Cruz Biotechnology, Santa-Cruz, CA), rabbit anti-CD2AP 1774 or 1764 (raised against mouse CD2AP as described [START_REF] Lehtonen | In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2[END_REF]), rabbit anti-phospho-Akt (Ser473) (Cell Signalling Technology, Danvers, MA), mouse anti-Pan Akt (R&D Systems, Minneapolis, MN), mouse anti-phosphotyrosine PT-66 (Sigma) or mouse antiactin (Sigma) and Alexa Fluor 680 (Invitrogen) or IRDye 800 (LI-COR) donkey antirabbit, anti-goat or anti-mouse IgG followed by detection and quantitation with an Odyssey Infrared Imager (LI-COR).

A c c e p t e d M a n u s c r i p t

Immunoperoxidase staining

Kidney samples were fixed with 10% formalin, dehydrated and embedded in paraffin.

Immunoperoxidase staining was performed with a VectaStain Elite kit (Vector Laboratories, Burlingame, CA). Sections were deparaffinized, antigen retrieval was done by boiling for 15 minutes in a microwave oven in 10 mM citric acid (pH 6,0), and endogenous peroxidase was inactivated by incubation in 0,5% H 2 O 2 in methanol for 30 min. Sections were blocked with CAS-block (Zymed, South San Francisco, CA) and incubated with anti-SHIP2 I-20 or anti-CD2AP 1774 diluted in ChemMate (Dako, Glostrup, Denmark) and with biotinylated rabbit anti-goat or goat anti-rabbit secondary antibody followed by incubation with ABC-reagent and AEC (Dako) colour development. Sections were counterstained with hematoxylin and mounted with Shandon Immu-Mount (Thermo Scientific, Waltham, MA). In db/db mice SHIP2 expression was rated (scale 1-5) by two independent researchers blinded from the genotypes. From each mouse (6 diabetic and 6 control), 8 glomeruli were photographed with a Nikon Eclipse 800 microscope with the same microscope settings for analysis.

Pull-down assay

Generation of glutathione-S-transferase (GST)-tag fusion protein constructs of mouse CD2AP 1.SH3, 2.SH3 and 3.SH3 domains has been previously described [START_REF] Palmen | Interaction of endogenous nephrin and CD2-associated protein in mouse epithelial M-1 cell line[END_REF]. The NH 2 -terminus of mouse CD2AP (aa 1-330) was subcloned into pGEX-4T-1 vector (Amersham Biosciences, Buckinghamshire, England) to produce GST-CD2AP-N.

GST and GST-CD2AP fusion proteins were produced in E. coli BL21(DE3) (Stratagene; La Jolla, CA) and purified on glutathione-Sepharose beads (Amersham Biosciences). Cell and glomerular lysates were precleared with glutathione-Sepharose beads at 4°C for 1 h, followed by incubation at 4°C for 4 h with GST-CD2AP fusion proteins or GST alone (20 µg each) immobilized on beads. Beads were washed extensively with lysis buffer, boiled in Laemmli sample buffer and proteins were separated by 10% SDS-PAGE for immunoblotting with anti-SHIP2 I-20.

A c c e p t e d M a n u s c r i p t

Immunoprecipitation

HIRc cells in basal state, starved 16 hours, or starved 16 hours and stimulated with 17 nM insulin for 7 minutes were lysed as described in 2.4.. Lysates were precleared with protein A-Sepharose beads (Invitrogen) or TrueBlot anti-goat Ig IP Beads (eBioscience, San Diego, CA) at 4°C for 1 h and incubated with anti-CD2AP 1764 [START_REF] Lehtonen | In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2[END_REF] or normal rabbit serum for 16-20 h at 4°C, or alternatively, with anti SHIP2 I-20 or goat IgG (5 μg). Immune complexes were bound to protein A-Sepharose or goat TrueBlot beads at 4°C for 1 h, washed 3 times with lysis buffer and boiled in Laemmli sample buffer for immunoblotting with anti-SHIP2 I-20, anti-CD2AP 1764, or antiphosphotyrosine.

Transient transfections and infections

Proliferating human podocytes were transfected with human SHIP2 cDNA with HA-tag in pCGN vector (SHIP2-pCGN) [START_REF] Dyson | The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin[END_REF] or empty pCGN using Lipofectamine 2000 (Invitrogen) and used for experiments 48 h after transfection. In differentiated podocytes SHIP2 was overexpressed by lentiviral infection. SHIP2 with HA-tag was subcloned from SHIP2-pCGN into pSIN18.cppt.hEF1αp.WPRE vector (Gropp et al 2003). Empty pSIN18.cppt.hEF1αp.WPRE vector was used as control. For virus production, KS pCMVΔ8.9 and pHCMV packaging plasmids, together with SHIP2 or control lentiviral vector, were transfected into HEK 293FT cells with Lipofectamine 2000. Medias were collected after 3 days, filtrated through 0.45 µm filter, and ultracentrifuged 85000 x g at + 4 °C for 90 min, to concentrate the viruses. The viruses were resuspended in PBS and added to differentiated podocytes on day 11 or 12 of differentiation. Podocytes were first incubated at +37 °C for 10 min, followed by centrifugation 1360 x g at +4 °C for 30 min. Virus-containing medium was replaced with regular medium 10-12 hours after the infection.

Immunofluorescence

Differentiated podocytes were starved for 14 hours, and stimulated with 200 nM insulin (Actrapid, Novo Nordisk, Denmark) for 5 or 15 min, and fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with CAS-block A c c e p t e d M a n u s c r i p t (Zymed). Cells were incubated with primary antibodies, rabbit anti-SHIP2 C76A7 (Cell Signalling Technology) or rabbit anti-CD2AP H-290 (Santa-Cruz) in ChemMate (Dako) for 1 h followed by secondary antibody Alexa Fluor 555 donkey anti-rabbit, in ChemMate for 1 h. Cells were mounted in Moviol and examined with a Zeiss Axiophot 2 microscope.

Akt activity measurements

Podocytes expressing SHIP2-pCGN or pCGN were serum and ITS starved overnight (18-

Fluorescence-activated cell sorting (FACS)

Apoptosis in SHIP2 overexpressing differentiated podocytes was measured on day 13 or 14 of differentiation, 48 hours after the lentiviral infection, by fluorescence-activated cell sorting (FACS) using Annexin V-FITC Apoptosis Kit (Pharmingen, BD, Franklin Lakes, NJ) and double staining with 7-Aminoactinomycin D (7-AAD) (BD). Cells were trypsinized, combined with detached cells in media, and centrifuged. Cell pellet was washed twice with cold PBS and resuspended in Annexin V Binding Buffer (BD) containing Annexin V-FITC and 7-AAD. Cells were incubated for 15 min at room temperature in dark, washed twice with cold PBS, centrifuged and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). After fixation, cells were washed once with PBS and centrifuged, and then analyzed for apoptosis using FACSAria (BD). Cells positive for Annexin V-FITC and negative for 7-AAD were regarded as apoptotic, positive for both as late apoptotic, positive for 7-AAD and negative for Annexin V-FITC as necrotic, and negative for both as viable.

A c c e p t e d M a n u s c r i p t

High-glucose treatment

Human podocytes were cultured at + 37 C for 14 days (the time of their differentiation) in RPMI medium containing 30 mM glucose, or mannitol resulting in the same osmolality as a control. Insulin stimulations were performed as described in 2.9. and cells lysed and immunoblotted as described in 2.4.

Results

CD2AP interacts with SHIP2 in glomeruli.

The NH 2 -terminus of the adaptor protein CD2AP contains three SH3 domains, which can bind to proline-rich sequence motifs and thus link CD2AP to various signal transduction pathways. To identify proteins that specifically bind to the SH3-domains of CD2AP in glomeruli we performed yeast two-hybrid screening of a rat glomerular library using the SHIP2 has previously been shown to be widely expressed in different tissues [START_REF] Schurmans | The mouse SHIP2 (Inppl1) gene: complementary DNA, genomic structure, promoter analysis, and gene expression in the embryo and adult mouse[END_REF] but its function in the kidney has not been studied. To confirm that SHIP2 is expressed in glomeruli, we isolated rat glomeruli and tubuli by the sieving technique, and immunoblotted the fractions with SHIP2 antibodies. SHIP2 was found to be expressed in both glomeruli and tubuli in the kidney whereas CD2AP was found predominantly in the glomerular fraction (Fig. 1A). Immunoperoxidase staining of mouse kidney sections indicated that SHIP2 localizes in glomeruli (Fig. 1B), and staining of adjacent sections

Confirmation of the interaction between CD2AP and SHIP2.

The COOH-terminal region of SHIP2 that was identified in the yeast two-hybrid screening contains a proline-rich domain that, based on the screening, mediates the interaction of SHIP2 with the NH 2 -terminus of CD2AP harboring the three SH3 domains.

To map which of the SH3-domains of CD2AP mediates the interaction, we performed pull-down assays on human podocyte cell lysates with GST-CD2AP 1., 2. and 3. SH3

domains and the GST-CD2AP-NH 2 -terminus. The 3. SH3-domain of CD2AP pulled down endogenous SHIP2 from podocyte lysate (Fig. 2A), and similar result was obtained when the pull-down assay was performed on rat glomerular lysates (Fig. 2B), further confirming the interaction between SHIP2 and CD2AP in glomeruli and suggesting that the binding is primarily mediated by the 3. SH3-domain of CD2AP.

To confirm the physiological interaction between CD2AP and SHIP2, we performed a co-immunoprecipitation assay from HIRc cells using CD2AP antibodies. Immunoblotting of the precipitates with SHIP2 antibodies showed that CD2AP co-immunoprecipitates SHIP2, confirming their interaction (Fig. 2C). The interaction was seen similarly in basal state, starved and insulin stimulated cells, indicating that the interaction is not dependent on insulin stimulation. Interestingly, two different forms of SHIP2 were detected in the cell lysate, but only the lower one was precipitated by CD2AP. Immunoprecipitation with SHIP2 antibodies from insulin stimulated HIRc cells and probing the same blot with anti-SHIP2 and anti-phosphotyrosine antibodies showed that only the upper band of SHIP2 is phosphorylated (Fig. 2D), proving that CD2AP binds to SHIP2 in its nontyrosine phosphorylated form.

Insulin stimulation induces SHIP2 translocation to plasma membrane.

In differentiated podocytes, endogenous SHIP2 was located predominantly in the cytoplasm in basal and starved state (Fig. 3A-B), but after insulin stimulation, it was A c c e p t e d M a n u s c r i p t partly translocated to plasma membrane (Fig. 3C-D). CD2AP was localized in cytoplasm and the localization did not change after insulin stimulation (Fig. 3E-F).

SHIP2 overexpression downregulates insulin signalling in podocytes.

Activation of the PI3K pathway leads to phosphorylation of Akt protein kinase. To study the regulative function of SHIP2 on this pathway in podocytes, we overexpressed SHIP2 in cultured podocytes, stimulated them with insulin and measured Akt phosphorylation level by immunoblotting. In unstimulated cells no difference in Akt phosphorylation was detected when SHIP2 overexpressing cells were compared with control cells transfected with an empty vector (Fig. 4A). But when podocytes were stimulated with insulin, Akt phosphorylation level remained 15.5% lower in SHIP2 overexpressing cells (Fig. 4B).

The downregulation of Akt activation was confirmed with an ELISA-based Akt activity assay, showing that in SHIP2 overexpressing podocytes Akt activity was not increased after insulin stimulation (Fig. 5A), in contrast to control cells (Fig. 5B), where it was increased by 32%. The difference in Akt activity between SHIP2 overexpressing and control podocytes after insulin stimulation was 35% (p=0.02, Student's t-test). Together, these results indicate that SHIP2 downregulates the insulin signalling pathway in podocytes.

SHIP2 overexpression promotes apoptosis in cultured podocytes.

Apoptosis is normally detected in low rates in differentiated podocytes. To study if SHIP2 affects apoptosis, SHIP2 was overexpressed in differentiated podocytes by lentiviral infection and apoptosis was measured by FACS. In control podocytes, infected with empty vector, the apoptosis rate was 4.5%, whereas SHIP2 overexpression increased the proportion of apoptotic cells to 8.6% (Fig. 6A-C). The proportion of necrotic cells was not increased by SHIP2 overexpression.

SHIP2 is upregulated in glomeruli of diabetic rats and mice.

SHIP2 expression in vivo in glomeruli of insulin resistant animals was studied using obese Zucker rats, that are insulin resistant and slightly diabetic due to a mutation in the leptin receptor gene [START_REF] Chua | Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor[END_REF]. Kidneys were analyzed at two time points, 12 and A c c e p t e d M a n u s c r i p t 40 weeks of age, 40 weeks being the age when proteinuria has been reported to start [START_REF] Coimbra | Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes[END_REF]. Kidneys from 12 weeks old rats appeared histologically normal with hematoxylin-eosin staining (data not shown), but immunoblot analysis of isolated glomeruli showed marked upregulation of SHIP2 expression in obese (fa/fa) rats when compared to lean controls (fa/+) (Fig. 7A,D). At 40 weeks of age the obese rats had slightly higher weights (data not shown) and blood glucose levels (table 1) than the controls, but these differences were not statistically significant. At this time point the obese rats also had proteinuria, as the urine albumin/creatinine ratio was higher in obese rats than in controls (table 1). At 40 weeks of age the elevation of SHIP2 levels in isolated glomeruli of obese rats continued (Fig. 7B,E), whereas in tubuli SHIP2 was expressed at equal levels (Fig. 7C,F).

As another animal model of insulin resistant diabetes we used db/db mice that also have a mutation in the leptin receptor gene [START_REF] Chua | Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor[END_REF]. These mice were highly hyperglycaemic at the age of 8 weeks (table 1) and clearly obese compared to heterozygous db/+ controls. They also had notable albuminuria (table 1). SHIP2 expression was studied by immunohistochemistry and the level of SHIP2 was graded in eight glomeruli from each sample by two independent researchers blinded from the genotypes. SHIP2 expression level was rated higher in podocytes in the diabetic group (Fig. 8), showing a similar trend to the obese rats, but there was some variation between individual mice and the difference did not reach statistical significance (p=0.059).

SHIP2 expression in cultured podocytes is not regulated by high glucose.

Cultured human podocytes were used to test whether SHIP2 is upregulated in a highglucose environment. Podocytes were cultured in cell culture medium containing high glucose concentration, 30 mmol/l, for the time of their differentiation (two weeks) or mannitol as an osmotic control. SHIP2 was expressed at equal levels in both conditions, and also the Akt phosphorylation response to insulin was similar (Fig. 9).

A c c e p t e d M a n u s c r i p t

Discussion

Podocytes have been shown to belong to insulin target cells [START_REF] Coward | The human glomerular podocyte is a novel target for insulin action[END_REF] and to develop insulin resistance (Tejada et al 2008), but the mechanisms have not been elucidated. Considering the findings from clinical studies linking insulin resistance to diabetic nephropathy [START_REF] Parvanova | Insulin resistance and microalbuminuria: a crosssectional, case-control study of 158 patients with type 2 diabetes and different degrees of urinary albumin excretion[END_REF][START_REF] Orchard | Nephropathy in type 1 diabetes: a manifestation of insulin resistance and multiple genetic susceptibilities? Further evidence from the Pittsburgh Epidemiology of Diabetes Complication Study[END_REF], it can be hypothesized that disturbances in insulin signalling affect podocyte function. This study identifies SHIP2 as a new interaction partner for CD2AP in glomeruli and focuses on its role in the regulation of insulin signalling in podocytes.

The interaction of CD2AP and SHIP2 was mapped by pull down assay to be mediated by the SH3 domains of CD2AP, most strongly by the third one, that are known to bind to proline rich domains. SHIP2 has a proline rich domain with the consensus sequence for SH3 domain ligand [START_REF] Marion | The gene INPPL1, encoding the lipid phosphatase SHIP2, is a candidate for type 2 diabetes in rat and man[END_REF], and interestingly, this domain was covered by both SHIP2 clones identified in the yeast-two-hybrid screening. Immunoprecipitation experiments confirmed that endogenous CD2AP and SHIP2 proteins are in the same complex, and that the interaction is not regulated by insulin stimulation. However, CD2AP was found to bind SHIP2 in its non-tyrosine phosphorylated form. SHIP2

tyrosine phosphorylation has previously shown to be induced by insulin and other growth factors [START_REF] Habib | Growth factors and insulin stimulate tyrosine phosphorylation of the 51C/SHIP2 protein[END_REF] and suggested to alter its subcellular localization and enzymatic activity [START_REF] Prasad | Specific tyrosine phosphorylations mediate signal-dependent stimulation of SHIP2 inositol phosphatase activity, while the SH2 domain confers an inhibitory effect to maintain the basal activity[END_REF]. Our results show that insulin stimulation induces partial SHIP2 translocation to plasma membrane in differentiated podocytes, a similar phenomenon as seen previously in COS-7 cells after EGF stimulation [START_REF] Pesesse | The Src homology 2 domain containing inositol 5phosphatase SHIP2 is recruited to the epidermal growth factor (EGF) receptor and dephosphorylates phosphatidylinositol 3,4,5-trisphosphate in EGF-stimulated COS-7 cells[END_REF][START_REF] Dyson | The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin[END_REF]. In that model, SHIP2 was also shown to regulate actin [START_REF] Dyson | The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin[END_REF]. Thus, alterations in SHIP2 expression in diabetes could affect the dynamic regulation of the actin cytoskeleton needed to maintain the functional structure of the podocyte foot processes. Interestingly, also CD2AP has been shown to participate in the regulation of actin organization [START_REF] Kirsch | CMS: an adapter molecule involved in cytoskeletal rearrangements[END_REF], and to function as a scaffolding protein that binds to membrane proteins [START_REF] Dustin | A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts[END_REF][START_REF] Palmen | Interaction of endogenous nephrin and CD2-associated protein in mouse epithelial M-1 cell line[END_REF], actin cytoskeleton [START_REF] Lehtonen | CD2-associated protein directly interacts with the actin cytoskeleton[END_REF], and signalling proteins [START_REF] Huber | Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKTdependent signaling[END_REF]. Our results suggest that CD2AP could have a role in the formation of signalling complexes Apoptosis rate was markedly increased in SHIP2 overexpressing differentiated podocytes, in which apoptosis is normally detected in low levels. Akt phosphorylation, also inducded by CD2AP, has previously shown to act as a survival signal in podocytes protecting them from apoptosis [START_REF] Huber | Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKTdependent signaling[END_REF], suggesting that impaired insulin response might contribute to podocyte loss in diabetic nephropathy. In addition to the regulation of PI3K pathway, SHIP2 also modulates, via its docking properties, JNK activation involved in apoptotic pathways [START_REF] Xie | The docking properties of SHIP2 influence both JIP1 tyrosine phosphorylation and JNK activity[END_REF], this being another possible mechanism by which SHIP2 could increase apoptosis.

In addition to the in vitro results, we found that SHIP2 is upregulated in vivo in glomeruli of obese Zucker rats and db/db mice, two well characterized models of insulin resistance and type II diabetes. In both of these models the development of proteinuria and renal injury has been analyzed, and they have been shown to share multiple similarities to early human diabetic nephropathy [START_REF] Coimbra | Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes[END_REF][START_REF] Sharma | Diabetic kidney disease in the db/db mouse[END_REF]. Our results show upregulation of SHIP2 in obese rat glomeruli already before histological changes and the reported age of onset of proteinuria. Thus SHIP2 upregulation is not a secondary effect of A c c e p t e d M a n u s c r i p t podocyte damage but rather might be involved in the development of diabetic nephropathy. Interestingly, SHIP2 was not upregulated in tubuli of obese Zucker rats, which further underlines the nature of podocytes as targets of insulin action and resistance.

Hyperglycaemia is known to induce insulin resistance in muscle cells by reducing Akt phosphorylation in response to insulin [START_REF] Tomas | Hyperglycemia and insulin resistance: possible mechanisms[END_REF]. This prompted us to study if high glucose affects insulin signalling in podocytes by regulating SHIP2 expression. Our findings did not support this hypothesis as high glucose concentration did not induce SHIP2 upregulation in cultured podocytes, nor did it affect Akt phosphorylation. We cannot exclude the possibility that SHIP2 upregulation in glomeruli in vivo could result from glucotoxicity, when the podocytes can be exposed to hyperglycaemia for a longer time. It seems more likely though, that upregulation of SHIP2 in vivo could be due to long term programming of cells either genetically or by environmental factors, possibly including obesity. Also our results from animal models suggest that upregulation of SHIP2 does not depend on hyperglycaemia, as SHIP2 was upregulated in obese Zucker rats, which did not have significantly elevated blood glucose levels. Both animal models have a mutation in the leptin receptor gene, leading to hyperphagia and obesity [START_REF] Chua | Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor[END_REF]. Many of the factors promoting insulin resistance, namely adipocytokines, free fatty acids, inflammation and oxidative stress, are linked to obesity [START_REF] Savage | Disordered lipid metabolism and the pathogenesis of insulin resistance[END_REF][START_REF] Houstis | Reactive oxygen species have a causal role in multiple forms of insulin resistance[END_REF]. Obesity is a central feature of the metabolic syndrome, and interestingly, SHIP2 gene INPPL1 polymorphisms have been associated to the components of the metabolic syndrome and type II diabetes in humans [START_REF] Marion | The gene INPPL1, encoding the lipid phosphatase SHIP2, is a candidate for type 2 diabetes in rat and man[END_REF][START_REF] Kaisaki | Polymorphisms in type II SH2 domaincontaining inositol 5-phosphatase (INPPL1, SHIP2) are associated with physiological abnormalities of the metabolic syndrome[END_REF].

In clinical studies, insulin resistance has been associated with proteinuria in both type 1 and type 2 diabetic patients [START_REF] Parvanova | Insulin resistance and microalbuminuria: a crosssectional, case-control study of 158 patients with type 2 diabetes and different degrees of urinary albumin excretion[END_REF][START_REF] Orchard | Nephropathy in type 1 diabetes: a manifestation of insulin resistance and multiple genetic susceptibilities? Further evidence from the Pittsburgh Epidemiology of Diabetes Complication Study[END_REF], and the insulin sensitizing agents thiazolidinediones have been shown to have a renoprotective effect in diabetic patients [START_REF] Miyazaki | Rosiglitazone decreases albuminuria in type 2 diabetic patients[END_REF] and animal models [START_REF] Zhang | Rosiglitazone reduces renal and plasma markers of oxidative injury and reverses urinary metabolite abnormalities in the amelioration of diabetic nephropathy[END_REF], but the mechanism is not known. Previously SHIP2 levels have been found to be elevated in db/db mouse muscle and adipose tissues [START_REF] Hori | Association of SH2-containing inositol phosphatase 2 with the insulin resistance of diabetic db/db mice[END_REF], and could be lowered by In conclusion, this study shows that glomerular podocytes express lipid phosphatase SHIP2 and that SHIP2 downregulates insulin response and promotes apoptosis in podocytes. Further, SHIP2 expression was found to be elevated in glomeruli of diabetic rats prior to the development of proteinuria. Collectively, these results demonstrate a possible mechanism for insulin resistance in podocytes and suggest the involvement of SHIP2 in altered podocyte function in diabetes. SH3 domain of CD2AP (CD2AP-N and CD2AP-2.SH3, lanes 1 and 3) but not with GST alone (lane 5). Lane 6, rat glomerular lysate. Human podocyte and rat glomerular lysates were incubated with GST-CD2AP fusion proteins or GST alone immobilized on beads.

Beads were washed, proteins separated by SDS-PAGE and immunoblotted with anti-SHIP2. (C) SHIP2 is co-immunoprecipitated with CD2AP antibodies (lanes 1, 4 and 7),

but not with control serum (lanes 2, 5 and 8), from HIRc cells in basal, starved or insulin stimulated state, indicating that the interaction is not dependent on insulin stimulation.

SHIP2 is seen as two bands in lysate (lanes 3, 6 and 9) but only the lower one is co-

  20 h) and stimulated with 200 nM insulin for 15 min. Akt phosphorylation was measured by immunoblotting with Phospho-Akt (Ser473) antibody recognizing Akt1 phosphorylated at Ser473 and Akt2 and Akt3 phosphorylated at corresponding residues, and compared to total Akt level detected with Pan Akt antibody. Akt kinase activity was measured with ELISA-based Akt/PKB Kinase Activity Assay Kit (Assay Designs, Enzo Biochem, New York, NY).

  NH 2 -terminus of CD2AP (aa 1-330) as a bait. Screening of 1x10 6 yeast transformants led to the identification of two SHIP2 clones covering amino acids 726-1257. SHIP2 is a 4)bisphosphate (PI(3,4)P 2 ), and thus negatively regulates the phosphoinositol 3-kinase (PI3K) pathway mediating various metabolic effects of insulin[START_REF] Dyson | The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2[END_REF].

  podocyte marker CD2AP confirmed SHIP2 expression in podocytes (Fig.1C-D).

  investigate the function of SHIP2 in podocytes we overexpressed it in cultured human podocytes and measured insulin sensitivity of the cells by measuring Akt phosphorylation and activation in response to insulin. SHIP2 overexpression downregulated the response like it has been previously shown to do in other insulin responsive cells[START_REF] Sasaoka | SH2-containing inositol phosphatase 2 negatively regulates insulin-induced glycogen synthesis in L6 myotubes[END_REF], Wada et al 2001). The mechanisms of insulin resistance seem to be partly shared and partly separate in different cell types, and have been mainly studied in the traditional insulin target cells, myocytes, adipocytes and hepatocytes[START_REF] Tomas | Hyperglycemia and insulin resistance: possible mechanisms[END_REF][START_REF] Savage | Disordered lipid metabolism and the pathogenesis of insulin resistance[END_REF][START_REF] Houstis | Reactive oxygen species have a causal role in multiple forms of insulin resistance[END_REF]. Podocyte cell line derived from db/db mice has earlier been shown to be insulin resistant compared to podocyte line from control mice(Tejada et al 2008). The results of this study identify the upregulation of SHIP2 as a mechanism inducing impaired insulin signalling in podocytes, leading to reduced Akt phosphorylation and activation.

  thiazolidinedione drug, rosiglitazone. Considering the previous data together with our finding of SHIP2 expression in glomerular podocytes in db/db mice, it could be speculated that SHIP2 might have a role in the effects of thiazolidinediones in the kidney.

Fig. 1 .

 1 FIGURE LEGENDS

Fig. 2 .

 2 Fig. 2. SHIP2 associates with CD2AP.(A) SHIP2 is pulled down from human podocyte cell lysate with GST-3. SH3 domain of CD2AP (CD2AP-3.SH3, lane 4) but not with GST alone (lane 5). Lane 6, podocyte cell lysate. (B) Also from rat glomerular lysate SHIP2 is pulled down with GST-3. SH3 domain of CD2AP (CD2AP-3.SH3, lane 4), and in addition, weaker bands are observed with GST-CD2AP NH 2 -terminus and GST-2.

  precipitated with CD2AP. (D) Immunoprecipitation with SHIP2 antibodies from HIRc cells stimulated with 17 nM insulin for 7 min, and probing the same blot with anti-SHIP2 and anti-phosphotyrosine antibodies shows that the upper band of SHIP2 is A c c e p t e d M a n u s c r i p t phosphorylated. Cells were lysed and immunoprecipitations performed with anti-CD2APand control serum, or anti-SHIP2 and control IgG, followed by immunoblotting with anti-SHIP2, anti-CD2AP and anti-phosphotyrosine.

Fig. 3 .

 3 Fig. 3. Insulin stimulation induces SHIP2 translocation to plasma membrane. (A-D) In differentiated podocytes, endogenous SHIP2 is localized mainly in the cytoplasm in basal state (A) and starved cells (B). After 5 minutes of insulin stimulation, SHIP2 is occasionally seen at the cell membrane (C, arrow), and after 15 minutes of stimulation the membrane localization becomes more frequent (D, arrows). (E-F) CD2AP is localized in cytoplasm in basal state (E), and its localization does not change after insulin stimulation (F). Bar, 40 µm.

Fig. 4 .Fig. 6 .

 46 Fig. 4. SHIP2 downregulates insulin signalling in podocytes. (A) In unstimulated SHIP2 overexpressing human podocytes the phosphorylation level of Akt does not differ from control cells transfected with an empty vector. (B) After 15 min insulin stimulation the phosphorylation level of Akt remained 15.5% lower in SHIP2 overexpressing podocytes than in control cells. SHIP2 was transiently transfected into proliferating podocytes, cells were starved overnight and stimulated with 200 nM insulin for 15 min, lysed and immunoblotted with anti-P-Akt (Ser473) and anti-Pan Akt. Quantitation was done with Odyssey Infrared Imager, and the amount of phosphorylated Akt was adjusted to total Akt in each lane. Bars (above) show results from either 8 experiments (4 independent experiments in duplicate) (A) or 12 experiments (4 independent experiments in triplicate) (B). Representative immunoblots are shown below. Akt phosphorylation level in SHIP2 overexpressing podocytes is presented as percentage of control. Bars show mean and error bars STDEV.  p<0.01, Wilcoxon signed rank test.

Fig. 7 .

 7 Fig. 7. SHIP2 is upregulated in obese Zucker rat glomeruli. (A-B) SHIP2 is upregulated in the glomeruli of 12 weeks (A) and 40 weeks (B) old obese Zucker rats when compared to lean controls. (C) In tubuli of 40 weeks old rats SHIP2 is expressed at equal levels in both groups. Glomeruli and tubuli were isolated, lysed and immunoblotted with anti-SHIP2 and anti-actin. SHIP2 expression was quantitated with Odyssey Infrared Imager and adjusted to actin in each lane. The bars (A-C) show the mean expression in arbitrary units (error bars STDEV). A, n=3. B and C, n =6.  p<0.05, student's t-test. (D) Immunoblot of SHIP2 in glomeruli of 12 weeks old Zucker rats. (E-F) Representative immunoblots of SHIP2 in glomeruli (E) and tubuli (F) of 40 weeks old rats.

Fig. 8 .

 8 Fig. 8. SHIP2 in diabetic mouse glomeruli. (A-B) SHIP2 staining in diabetic db/db mouse glomerular podocytes (B) compared to db/+ control (A). 8-week old db/+ and db/db mouse kidney paraffin sections were processed for immunoperoxidase staining and labelled with anti-SHIP2. Bar, 20 µm. (C) SHIP2 expression was rated visually (scale 1-5) by two independent researchers blinded from the genotypes. Analysis of 6 db/+ and 6

Fig. 9 .

 9 Fig. 9. SHIP2 expression in cultured podocytes is not regulated by high glucose. (A) High-glucose treatment does not induce SHIP2 upregulation in cultured human podocytes compared to mannitol treated cells. (B-C) The level of Akt phosphorylation was the same in high-glucose and mannitol treated cells both without (B) and with (C) insulin stimulation. Podocytes were cultured for two weeks in RPMI medium containing 30 mM glucose, or mannitol resulting in the same osmolality as a control. Quantitation of the immunoblots was performed with Odyssey Infrared Imager, and SHIP2 expression was adjusted to actin, and P-Akt to total Akt in each lane. The bars (A-C) show the mean results of 6 experiments (3 independent experiments done in duplicate) in arbitrary units (error bars STDEV). (D) A representative immunoblot of SHIP2 in high-glucose and mannitol treated podocytes. (E-F) Representative immunoblots of P-Akt in high-glucose and mannitol treated podocytes without (E) and with (F) insulin stimulation.

  Figure 1

  Figure 2

  Figure 4

  Figure 6

Table 1 . Blood glucose and urinary albumin values of Zucker rats and db/db mice.

 1 Blood glucose, mmol/l (n=6) Urine albumin to creatinine,

	mg/mg

Table 1 .

 1 Obese Zucker rats at 40 weeks had slightly higher blood glucose than controls, but this was not significant. The urinary albumin to creatinine ratio was elevated in the obese group,  p<0.05. Diabetic db/db mice had significantly higher blood glucose than controls and also albuminuria ( p<0.005) at the age of 8 weeks.

Acknowledgements

HIRc cells were kindly provided by professor Olefsky (UCSD, San Diego, USA), human SHIP2 cDNA with HA-tag in pCGN vector by professor Mitchell (Monash University, Clayton, Victoria, Australia), and pSIN18.cppt.hEF1αp.WPRE lentiviral vector by professor Otonkoski (University of Helsinki, Helsinki, Finland). This study was financially supported by the Academy of Finland, the European Research Council (242820), the Diabetes Research Foundation, the Foundation for Paediatric Research, Kyllikki and Uolevi Lehikoinen Foundation, and National Graduate School of Clinical Investigation (M.E.H). We thank Ulla Kiiski, Marcel Messing and Niina Ruoho for skilful technical assistance, and the Paediatric Graduate School of the University of Helsinki for support.

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