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Impact of Src Homology 2-Containing Inositol 5'-Phosphatase 2 Gene Polymorphisms Detected in a Japanese Population on Insulin Signaling

Department of Clinical Pharmacology (S.K., T.S., S.Y., H.T., S.K., I.K.) and First Department of Internal Medicine (S.M., K.F., T.W., M.K.), Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan; and Sainou Hospital (H.I.), Toyama 930-0887, Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., Department of Clinical Pharmacology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: tsasaoka-tym{at}umin.ac.jp.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Src homology 2-containing 5'-inositol phosphatase 2 (SHIP2) is known to be one of lipid phosphatases converting PI(3,4,5)P₃ to PI(3,4)P₂ in the negative regulation of insulin signaling with the fundamental impact on the state of insulin resistance. To clarify the possible involvement of SHIP2 in the pathogenesis of human type 2 diabetes, we examined the relation of human SHIP2 gene polymorphisms to type 2 diabetes in a Japanese population. We identified 10 polymorphisms including four missense mutations. Among them, single nucleotide polymorphism (SNP)3 (L632I) was located in the 5'-phosphatase catalytic region, and SNP5 (N982S) was adjacent to the phosphotyrosine binding domain binding consensus motif in the C terminus. SNP3 was found more frequently in control subjects than in type 2 diabetic patients, suggesting that this mutation might protect from insulin resistance. Transfection study showed that expression of SNP3-SHIP2 inhibited insulin-induced PI(3,4,5)P₃ production and Akt2 phosphorylation less potently than expression of wild-type SHIP2 in CHO-IR cells. Insulin-induced tyrosine phosphorylation of SNP5-SHIP2 was decreased compared with that of wild-type SHIP2, resulting in increased Shc/Grb2 association and MAPK activation. These results indicate that the polymorphisms of SHIP2 are implicated, at least in part, in type 2 diabetes, possibly by affecting the metabolic and/or mitogenic insulin signaling in the Japanese population

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
TYPE 2 (NONINSULIN DEPENDENT) diabetes mellitus is a disorder characterized by insulin resistance in the target tissue of insulin with insufficient insulin secretion in pancreatic ß-cells (1). The molecular mechanisms underlying the insulin resistance in insulin signals leading to glucose uptake have been intensively investigated (1, 2). After insulin stimulation, activated insulin receptors phosphorylate tyrosine residues of insulin receptor substrates (1, 2, 3). The phosphorylated insulin receptor substrate binds to the p85 subunit of phosphatidylinositol (PI)3-kinase and activates its p110 catalytic subunit (1, 4). The activated PI3-kinase functions as a lipid kinase that phosphorylates the D-3 position of the phosphoinositide ring and produces PI(3,4,5)-trisphosphate [PI(3,4,5)P₃] (2, 4). PI(3,4,5)P₃ functions as a lipid second messenger in the activation of downstream signaling of PI3 kinase including Akt/protein kinase B and atypical protein kinase C (5, 6, 7, 8, 9). This series of insulin signaling leads to glucose uptake by promoting GLUT4 translocation from the cytosol to the plasma membrane (1, 2, 5). Because the PI3 kinase pathway plays a crucial role in the biological effects of insulin (6, 7), it is possible that the attenuation of the PI3 kinase-mediated insulin signaling is associated with the insulin resistance in type 2 diabetes.

Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) was identified as a 5'-lipid phosphatase responsible for the regulation of insulin signaling by hydrolyzing PI3-kinase product PI(3,4,5)P₃ to PI(3,4)-bisphosphate (10, 11). Overexpression of SHIP and SHIP2 inhibited insulin-induced glucose uptake and glycogen synthesis in 3T3-L1 adipocytes and L6 myotubes (12, 13, 14). Targeted disruption of SHIP2 gene in mice caused an increase in insulin sensitivity without affecting other biological systems based on the histological analyses (15). Expression of SHIP2 was greatly increased in the skeletal muscle and fat tissue of diabetic db/db mice (16). These reports suggest that SHIP2 is a physiologically important negative regulator relatively specific to insulin signaling with an impact on the state of insulin resistance. In addition, SHIP2 is in human chromosome 11q13-14, which is suggested to be linked to type 2 diabetes with insulin resistance and hypertension (17, 18, 19). Therefore, it is possible that SHIP2 is involved in the pathogenesis of insulin resistance of type 2 diabetes mellitus in humans (20). In fact, a recent report (21) has shown that some polymorphisms of SHIP2 found in British and French type 2 diabetes are associated with metabolic syndrome including type 2 diabetes and hypertension. However, the molecular mechanism by which the identified mutant SHIP2 is implicated in the state of insulin resistance is unknown. Furthermore, Japanese type 2 diabetes appears to possess a different genetic background based on clinically not being particularly obese compared with North American and European Caucasians (21). Thus, it would be of particular importance to clarify the involvement of SHIP2 in different ethnic groups.

In the present study, we investigated a relation between single nucleotide polymorphisms (SNPs) on human SHIP2 gene and the pathogenesis of type 2 diabetes mellitus in a Japanese population. Furthermore, to examine the impact of the SNPs on insulin signaling, SHIP2 mutants were expressed in CHO cells overexpressing insulin receptors.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

We examined 106 unrelated Japanese subjects with type 2 diabetes diagnosed by World Health Organization criteria (22) and 100 nondiabetic Japanese control subjects without a family history of diabetes for the analysis of SNPs in SHIP2 gene. The clinical characteristics of all subjects are shown in Table 1. All type 2 diabetic patients were recruited from the outpatient clinic of the First Department of Internal Medicine, Toyama Medical and Pharmaceutical University Hospital (Toyama, Japan). Before participation, the purpose and risks of this study were precisely explained both orally and in writing, and written informed consent was obtained from all subjects. The protocol of this study was approved by ethics committee at Toyama Medical and Pharmaceutical University.

Human crystal insulin was provided by Novo Nordisk Pharma Ltd. (Copenhagen, Denmark). A monoclonal anti-FLAG antibody (M2) and a polyclonal anti-FLAG antibody were purchased from Sigma (St. Louis, MO). A monoclonal anti-Shc antibody, a polyclonal anti-Shc antibody, and a monoclonal anti-Grb2 antibody were from Transduction Laboratories, Inc. (Lexington, KY). A monoclonal anti-Akt1 antibody and a monoclonal anti-phosphotyrosine antibody (PY99) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A polyclonal anti-Akt2 antibody was from Calbiochem, Inc. (San Diego, CA). A polyclonal anti-Ser⁴⁷³ phosphospecific Akt antibody, a polyclonal anti-p44/42 MAPK antibody, and a polyclonal antiphospho p44/42 MAPK antibody were from Cell Signaling Technology, Inc. (Beverly, MA). A monoclonal anti-PI(3,4,5)P₃ antibody was from Echelon Biosciences Inc. (Salt Lake City, UT). A biotinylated goat antimouse IgM was from Jackson ImmunoResearch (West Grove, PA). A streptavidin-AlexaFluor 488 was from Molecular Probes (Eugene, OR). All other routine reagents were analytical grade and purchased from Sigma or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Identification of SNPs in SHIP2 gene

Blood samples were obtained from all subjects, and genomic DNA was extracted from peripheral leukocytes using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). PCR and direct sequencing were performed to identify SNPs in SHIP2 DNA. As a primary study, we examined genomic DNAs from 30 Japanese type 2 diabetic patients and 30 Japanese nondiabetic control subjects. PCR primers used for amplification of all 28 exons and exon-intron boundary of SHIP2 gene are shown in the supplemental data (1) (published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org/), which were based on the sequence of SHIP2 gene reported in DDBJ/EMBL/GenBank (accession no. NT_033927). Because exon 26 sequence is extremely long to amplify with single PCR, exon 26 was divided into two segments. For amplification of exons 1, 4, 26, and 27, PCR was carried out in a 50-µl mixture containing 200 ng template DNA, 200 nM each primer, 2.5 mM MgCl₂, 200 nM each dNTP, and 0.25 U LaTaq DNA polymerase with 1x PCR buffer supplied by the manufacturer (Takara, Tokyo, Japan). The PCR conditions were the following: initial denaturation at 94 C for 5 min, 30 cycles of denaturation at 94 C for 20 sec, annealing and elongation at 68 C for 1 min, and final elongation at 68 C for 10 min. For amplification of other exons, PCR was carried out in 50 µl mixture containing 200 ng template DNA, 200 nM each primer, 2.5 mM MgCl₂, 200 nM each dNTP, and 1.0 U KOD plus DNA polymerase with 1x PCR buffer supplied by the manufacturer (Toyobo, Osaka, Japan). The PCR conditions were the following: initial denaturation at 94 C for 2 min, 35 cycles of denaturation at 94 C for 15 sec, annealing, and elongation at 68 C for 40 sec. The PCR products were purified with MinElute PCR purification kit (QIAGEN, Madison, WI), and SNPs in SHIP2 human genomic DNA were identified by direct sequence with the ABI PRISM 377 sequencer, BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (PE Applied Biosystems, Foster City, CA).

Genotyping of SNPs in the SHIP2 gene

In the association study, genotypes of all 106 unrelated type 2 diabetic patients and 100 unrelated nondiabetic control subjects including the subjects with primary studies were assessed by the PCR-restriction fragment length polymorphism method (23). The identified SNPs except SNP1 altered recognition sites of the following endonucleases: Tth111I (Promega) for SNP2, DdeI (Toyobo) for SNP3 and SNP6, NlaIII (New England Biolabs Inc, Beverly, MA) for SNP4, MaeIII (Roche, Penzberg, Germany) for SNP5, BsmI (New England Biolabs) for SNP7, PstI (Toyobo) for SNP8, BslI (New England Biolabs) for SNP9, and Tsp45I (New England Biolabs) for SNP10. To detect each genotype, PCR products were digested with endonucleases, and these fragments were resolved in 8% polyacrylamide gel and stained with ethidium bromide (Nippon Gene, Tokyo, Japan). SNP1 genotyping was performed by direct sequencing.

Construction of expression vectors

A fragment of human SHIP2 (hSHIP2) cDNA (Human Liver Marathon-Ready cDNA, Clontech, Palo Alto, CA), which includes the whole coding region without possessing the termination codon, was constructed with a 5' primer containing a HindIII restriction site (underlined), 5'-AAGCTTGGTGCTGAGCCCTGCGCGGGCCATGGCCTCG-3'; and a 3'-primer containing an SalI restriction site (underlined), 5'-GTCGACAACTTGCTGAGCTGCAGGGTGTCCAGAAGG-3' based on the sequence reported in DDBJ/EMBL/GenBank (accession no. Y14385). The SHIP2 cDNA was ligated into the multiple cloning site of C-terminally FLAG-tagged mammalian expression vector pFLAG-CMV-5b (Sigma). We used primers described below for the expression of hSHIP2 containing various types of polymorphisms: SNP3-hSHIP2-flag 5'-GCAGGAAAGAGTTTGAGCCCCTAATCAGGGTGGACCAGCTCAACC-3' (sense) and 5'-GGTTGAGCTGGTCCACCCTGATTAGGGGCTCAAACTCTTTCCTGC-3' (antisense), SNP4-hSHIP2-flag 5'-CGTCACCAGCGACCATTCCCCCATGTTTGGGACATTTGAGGTTGG-3' (sense) and 5'-CCAACCTCAAATGTCCCAAACATGGGGGAATGGTCGCTGGTGACG-3' (antisense), SNP5-hSHIP2-flag 5'-CCCACCCAAGAACAGCTTCAGTAACCCTGCCTACTACGTCC-3' (sense) and 5'-GGACGTAGTAGGCAGGGTTACTGAAGCTGTTCTTGGGTGGG-3' (antisense), and SNP6-hSHIP2-flag 5'-GTGGTCCGGGGCCGTGGTGGGGGTGAGGCCCGTGGCCCACCACC-3' (sense) and 5'-GGTGGTGGGCCACGGGCCTCACCCCCACCACGGCCCCGGACCAC-3' (antisense). Identified polymorphisms were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with prepared primers. All of the expression constructs were confirmed by direct sequencing.

Culture of CHO-IR cells and plasmid transfection

Chinese hamster ovary cells overexpressing human insulin receptors (CHO-IR) were kindly supplied by Dr. Wataru Ogawa (Kobe University, Kobe, Japan) and were maintained in Ham’s F-12 medium containing 10% fetal calf serum (24). Human SHIP2 expression vectors were transfected into CHO-IR cells with TransIT-LT1 transfection reagent (Pan Vera Co., Madison, WI) according to the manufacturer’s instruction.

Immunoprecipitation and immunoblot analyses

Cells transfected with various mutants of SHIP2 were serum starved for 16 h and then stimulated with insulin for indicated times. The cells were lysed in a buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 1 mM Na₃VO₄, 160 mM sodium fluoride, 10 µg aprotinin/ml, and 10 µM leupeptin (pH 7.4) for 15 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. For immunoprecipitation analyses, the cell lysates were immunoprecipitated with indicated antibodies. The precipitants or supernatants were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween [10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20 (pH 7.4)] or with 2.5% BSA (Sigma) in Tris-buffered saline with Tween and then incubated at 4 C for 16 h with various antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) at 25 C for 1 h, followed by chemiluminescence detection using the enhanced chemiluminescence reagent according to the manufacturer’s instruction (Amersham Pharmacia Biotech).

Immunocytochemical detection of PI(3,4,5)P₃

Cells transfected with various mutants of SHIP2 grown on coverslips were serum starved for 16 h and then stimulated with insulin for 5 min. The cells were fixed with in 4% paraformaldehyde in Tris-buffered saline (TBS) at 25 C for 10 min. After being washed three times with TBS, the cells were permeabilized with 0.5% Triton X-100 at 25 C for 15 min and blocked with 10% goat serum in TBS at 37 C for 30 min. The cells were incubated with mouse anti-PI(3,4,5)P₃ IgM at a 1:100 dilution at 25 C for 1 h. After the cells were washed three times with 10% goat serum in TBS, biotinylated goat antimouse IgM was added for 30 min. After being washed, the cells were incubated with Streptavidin-AlexaFluor 488 in TBS for 30 min according to the manufacturer’s instruction (Echelon Biosciences Inc.). After the coverslips were mounted, intensity of the PI(3,4,5)P₃ fluorescence in the cells was analyzed with a confocal laser fluorescence inverted microscope (LSM510, Carl Zeiss, Inc., Oberkochen, Germany) (25).

Statistical analyses

² Analysis and Fisher’s exact test were conducted to analyze the differences of genotype and allele between type 2 diabetic patients and control subjects. The odds ratio, 95% confidence interval (CI), and the significance of the odds ratio were determined by logistic regression analyses. P < 0.05 was considered statistically significant by using statistical software STAT VIEW J-4.5 (Abacus Concepts Inc., Berkeley, CA). To estimate the linkage disequilibrium (LD) for all possible pair of SNPs, the conventional pair-wise LD coefficient (|D'| and ²) was calculated by means of expectation-maximization algorithm using Arlequin software version 2.00 (26). The significance in immunoblot analyses was determined by one-way ANOVA, followed by Scheffé multiple range test, using STAT VIEW.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Identification of 10 polymorphisms and their characteristics in human SHIP2 gene

We identified 10 polymorphisms in the exon and exon-intron boundary of SHIP2 gene in a primary study with genomic DNA from 30 type 2 diabetic patients and 30 nondiabetic control subjects in a Japanese population. One of the polymorphisms was located in 5'-untranslated region (UTR) of exon 1 (SNP1), which replaced G to C at position –74. Five polymorphisms were found in SHIP2 coding region, and three polymorphisms in the coding region (SNPs 2–4) were identified in the 5'-phosphatase region. SNP2 was a silent mutation in the exon 12 that replaced C to T at position 1368 of SHIP2 cDNA as already reported in DDBJ/EMBL/GenBank (accession no. Y14385). SNP3 was in the exon 16 that replaced CC to AA at position 1893 and 1894 of SHIP2 cDNA, which led to amino acid substitution from leucine to isoleucine at position 632 (L632I). SNP4 was identified in the exon 19 that replaced G to A at position 2161 of SHIP2 cDNA, which resulted in amino acid substitution from valine to methionine at position 721 (V721M). Concerning another two polymorphisms, SNP5 was seen in exon 26 that replaced A to G at position 2945 of SHIP2 cDNA, which led to amino acid substitution from asparagine to serine at position 982 (N982S). Interestingly, SNP5 located adjacent to the NPXY motif with phosphotyrosine binding (PTB) domain binding consensus. SNP6 was also identified in the exon 26 that replaced C to G at position 3248 of SHIP2 cDNA, which led to amino acid substitution from alanine to glycine at position 1083 (A1083G). The remaining four polymorphisms (SNPs 7–10) were identified as the intronic variants. SNP7 was in the intron 7 [intronic variant sequence (IVS) 6 + 31] that replaced G to A. SNP8 was in the intron 18 (IVS 17 + 35) that replaced C to T. SNP9 was in the intron 23 (IVS 23-6) that replaced T to C, and SNP10 was in the intron 27 (IVS 27-22) that replaced C to G. The positions of all identified polymorphisms in SHIP2 structure including the SH2 domain, 5'-phosphatase catalytic region, and PTB domain binding consensus motif are shown in Fig. 1.

View larger version (15K): [in this window] [in a new window]   FIG. 1. The location of identified 10 SNPs in human SHIP2 gene. The human genomic DNA of SHIP2 (Inppl1) was referred to in DDBJ/EMBL/GenBank (accession no. NT_033927). Black box, 5'- and 3'-UTR. Gray box, Coding region of 5'-phosphatase catalytic region. Deep gray box, Coding region of SH2 domain. Dotted line, Coding region of PTB domain binding consensus motif. Arrows, Location of identified 10 SNPs and the change of sequences in human SHIP2 gene.

We concomitantly detected SNPs 1–3 and 8 in the SHIP2 gene from the same subject in primary studies. Therefore, we calculated the coefficient |D'| and ² using expectation-maximization algorithm to reveal whether any combinations of SNPs are identified as LD block and haplotype in all subjects. The results of the pair-wise LD analyses were shown in supplemental data 2, and the distribution of |D'| value was illustrated in supplemental data 3. We found that the combination of SNP1-SNP2-SNP3 represented complete LD (|D'| = 1, ² = 1, P < 0.001). The mutant allele combination (composed of SNPs 1–3) and the mutant allele of SNP8 were less in diabetic patients than those in control subjects (P < 0.05). In addition, the multiple haplotype block was related to the low risk of type 2 diabetes according to logistic regression analyses (odds ratio, 0.371; 95% CI, 0.146–0.945; P < 0.05). All of SNPs including the multiple haplotype association were in Hardy-Weinberg equilibrium.

Effect of various SHIP2 expressions on insulin-induced production of PI(3,4,5)P₃ and phosphorylation of Akt

We next examined the impact of identified SHIP2 polymorphisms on insulin signaling in vitro. SHIP2 functions mainly via the 5'-phosphatase activity hydrolyzing the PI3-kinase product PI(3,4,5)P₃ in the negative regulation of insulin’s metabolic signaling (12). We examined the effect of various SHIP2 mutant expressions on the levels of PI(3,4,5)P₃. Insulin treatment increased the PI(3,4,5)P₃ levels analyzed with immunocytochemistry. Insulin-induced production of PI(3,4,5)P₃ was decreased by 65.9 ± 5.2% in wild-type (WT)-hSHIP2-expressing cells. The degree of the inhibition was apparently less in SNP3-hSHIP2-expressing cells compared with that in WT-hSHIP2-expressing cells. Expression of SNP3-hSHIP2 inhibited the production of PI(3,4,5)P₃ only by 26.3 ± 4.6%. The levels of PI(3,4,5)P₃ were less potently inhibited in the cells expressing SNP5-hSHIP2 and SNP6-hSHIP2 compared with WT-hSHIP2, whereas expression of SNP4-hSHIP2 effectively inhibited the PI(3,4,5)P₃ levels (Fig. 2). Akt is known to be a target molecule of PI3-kinase important for the metabolic action of insulin (27). Although Akt is composed of three isoforms, Akt1 and Akt2 are the major isoforms in CHO cells (28). In addition, Akt2 rather than Akt1 appears to be important for the subsequent metabolic signaling of insulin (29, 30). We examined the effect of various SHIP2 mutant expression on insulin-induced phosphorylation of Akt1 and Akt2. To investigate the effect of SHIP2 expression on phosphorylation of Akt1, the obtained cell lysates were immunoprecipitated with anti-Akt1-specific antibody. As can be seen in Fig. 3A, insulin induced phosphorylation of Akt1 at Ser⁴⁷³ in CHO-IR cells. The degree of Akt1 phosphorylation was not altered by expression of any SHIP2 mutants tested. Protein amounts in the anti-Akt1 precipitates among the samples were confirmed to be equal by immunoblotting with anti-Akt1 antibody (Fig. 3C). On the other hand, we employed the immunodepletion approach of Akt1 because Akt2 antibody useful for the effective immunoprecipitation was not available. Akt1 in the cell lysates was effectively immunoprecipitated by using anti-Akt1 antibody. The remaining supernatants did not contain a detectable amount of Akt1 (Fig. 3F), whereas the sample contained substantial amounts of Akt2 (Fig. 3D). Thus, the experimental condition is adequate for evaluating the effect of various SHIP2 mutants on insulin-induced phosphorylation of Akt2. Compared with insulin-induced phosphorylation of Akt2 in CHO-IR cells transfected with vacant vector, the phosphorylation level of Akt2 was reduced by 38.9 ± 0.7% in WT-hSHIP2-expressing cells (Fig. 3B). Interestingly, the degree of the inhibition was significantly less in SNP3-hSHIP2-expressing cells than that in WT-hSHIP2-expressing cells. Expression of SNP3-hSHIP2 inhibited the phosphorylation of Akt2 only by 9.3 ± 1.4%. Similar results of the Akt2 phosphorylation were obtained in 3T3-L1 adipocytes transfected with WT-hSHIP2 and SNP3-hSHIP2 by electroporation with the use of a Nucleofector (Amaxa, Cologne, Germany) (data not shown). In addition, the insulin-induced phosphorylation of Akt2 was less potently inhibited in CHO cells expressing SNP5-hSHIP2 and SNP6-hSHIP2 compared with WT-hSHIP2, whereas expression of SNP4-hSHIP2 effectively inhibited the Akt2 phosphorylation. Expression levels of these SHIP2 mutants were confirmed to be equal by immunoblotting the cell lysates with anti-FLAG antibody (Fig. 3E).

View larger version (22K): [in this window] [in a new window]   FIG. 2. Effect of SNPs 3–6-hSHIP2 expression on insulin-induced production of PI(3,4,5)P3. CHO-IR cells transfected with vacant vector (Mock), WT-hSHIP2 (WT), SNP3-hSHIP2 (SNP3), SNP4-hSHIP2 (SNP4), SNP5-hSHIP2 (SNP5), and SNP6-hSHIP2 (SNP6) were serum starved for 16 h and treated with 10 nM insulin for 5 min. The cells were fixed with 4% paraformaldehyde, immunostained with anti-PI(3,4,5)P3 antibody, and visualized by confocal laser fluorescence microscopy. The intensity of PI(3,4,5)P3 in the cells was determined by analyzing at least 100 cells at each point. Results are represented by mean ± SE of three separate experiments. *, P < 0.05 vs. Mock by one-way ANOVA with Scheffé test.

View larger version (49K): [in this window] [in a new window]   FIG. 3. Effect of SNPs 3–6-hSHIP2 expression on insulin-induced phosphorylation of Akt1 and Akt2. CHO-IR cells transfected with vacant vector (Mock), WT-hSHIP2 (WT), SNP3-hSHIP2 (SNP3), SNP4-hSHIP2 (SNP4), SNP5-hSHIP2 (SNP5), and SNP6-hSHIP2 (SNP6) were serum starved for 16 h and treated with 10 nM insulin for 5 min. Total cell lysates were immunoprecipitated with anti-Akt1 antibody. The precipitates were separated by SDS-PAGE and immunoblotted with anti-phospho-specific Akt antibody (A) or anti-Akt1 antibody (C). The anti-Akt1 supernatants were separated by SDS-PAGE and immunoblotted with anti-phospho-specific Akt antibody (B), anti-Akt1 antibody (F), or anti-Akt2 antibody (D). The total cell lysates were separated by SDS-PAGE and immunoblotted with anti-FLAG antibody (E). The amount of phosphorylated Akt1 and Akt2 was quantitated by densitometry. The intensity of the band derived from insulin-treated Akt2 phosphorylation in WT-hSHIP2 expressing cells was assigned a value of 1 arbitrary unit, and intensity of all treated groups was expressed as a fold value of control. Results are represented by mean ± SE of three separate experiments. *, P < 0.05 vs. Mock by one-way ANOVA with Scheffé test.

We further investigated the impact of identified SHIP2 polymorphisms on insulin’s mitogenic signaling in vitro. Insulin stimulates tyrosine phosphorylation of SHIP2NPXY⁹⁸⁶) and SHIP2 associates, via the phosphorylation site, with PTB domain of Shc (11). Thus, SHIP2 association with Shc is known to be abrogated by a mutant NPXF-rat SHIP2 (31). SNP5-hSHIP2 was a rare mutant found in one type 2 diabetic patient, and it was not statistically associated with type 2 diabetes. However, because SNP5-hSHIP2 encodes a mutant SHIP2 carrying amino acid substitution N982S adjacent to the NPXY motif of SHIP2, one can speculate that the amino acid substitution affects the tyrosine phosphorylation of SHIP2 (32). Therefore, we performed transient expression experiments to examine the effect of the substitution on insulin-induced tyrosine phosphorylation of SHIP2 in CHO-IR cells. As can be seen in Fig. 4, insulin induced a marked tyrosine phosphorylation of WT-hSHIP2. The degree of insulin-induced tyrosine phosphorylation of SNP5-hSHIP2 was significantly less than that of WT-hSHIP2. In contrast, the extent of tyrosine phosphorylation was comparable among SNP3-, SNP4-, SNP6-, and WT-hSHIP2. The tyrosine phosphorylation of SHIP2 is required for association with Shc (31). As shown in Fig. 5A, insulin induced association of Shc with tyrosine-phosphorylated SHIP2. Overexpression of WT-hSHIP2 increased the amount of tyrosine-phosphorylated SHIP2 associated with Shc. The degree of the amount of association was decreased by overexpression of SNP5-hSHIP2 compared with that by overexpression of WT-hSHIP2. Shc is also known to associate with Grb2 for the activation of MAPK in the mitogenic signaling of insulin (4, 33). Because SHIP2 association with Shc can compete for Shc/Grb2 binding (11), we examined the effect of WT-hSHIP2 and SNP5-hSHIP2 expression on insulin-induced Shc/Grb2 association (Fig. 5B). Insulin-induced Shc/Grb2 association was decreased by overexpression of WT-hSHIP2. The amount of Shc associated with Grb2 was greater in SNP5-hSHIP2-expressing cells than that in WT-hSHIP2-expressing cells. The amount of protein loaded among the sample was confirmed to be equal by immunoblot analysis with anti-Shc antibody (Fig. 5C). In accordance with the results of Shc/Grb2 association, expression of SNP5-hSHIP2 inhibited insulin-induced phosphorylation of p44/42 MAPK less potently than expression of WT-hSHIP2 (Fig. 5D). Similar protein levels loaded among the samples were confirmed by immunoblotting with anti-p44/42 MAPK antibody (Fig. 5E), and similar expression levels of WT-hSHIP2 and SNP5-hSHIP2 were assured by immunoblotting with anti-FLAG antibody (Fig. 5F). On the other hand, insulin-induced Shc/SHIP2 association, Shc/Grb2 association, and MAPK phosphorylation were similar among SNP3-, SNP4-, SNP6-, and WT-hSHIP2 (data not shown).

View larger version (31K): [in this window] [in a new window]   FIG. 4. Comparison of insulin-induced tyrosine phosphorylation among WT-, SNP3-, SNP4-, SNP5-, and 6-hSHIP2. CHO-IR cells transfected with vacant vector (Mock), WT-hSHIP2 (WT), SNP3-hSHIP2 (SNP3), SNP4-hSHIP2 (SNP4), SNP5-hSHIP2 (SNP5), and SNP6-hSHIP2 (SNP6) were serum starved for 16 h and treated with 50 nM insulin for 5 min. Total cell lysates were immunoprecipitated with anti-FLAG antibody. The precipitates were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (A) or anti-FLAG antibody (B). The amount of tyrosine phosphorylated SHIP2 was quantitated by densitometry. The intensity of the band derived from insulin-treated WT-hSHIP2-expressing cells was assigned a value of 1 arbitrary unit, and intensity of all treated groups was expressed as a fold value of control. Results are represented by mean ± SE of three separate experiments. *, P < 0.05 vs. WT-hSHIP2 by one-way ANOVA with Scheffé test.

View larger version (41K): [in this window] [in a new window]   FIG. 5. Effect of SNP5-hSHIP2 expression on insulin-induced Shc/SHIP2 association, Shc/Grb2 association, and MAPK phosphorylation. CHO-IR cells transfected with vacant vector (Mock), WT-hSHIP2 (WT), and SNP5-hSHIP2 (SNP5) were serum starved for 16 h and treated with 50 nM insulin for 5 min. Total cell lysates were immunoprecipitated with anti-Shc antibody. The precipitates were separated by SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody (A), anti-Grb2 antibody (B), or anti-Shc antibody (C). The total cell lysates were separated by SDS-PAGE and immunoblotted with antiphospho p44/42 MAPK antibody (D), anti-p44/42 MAPK antibody (E), or anti-FLAG antibody (F). The amount of SHIP2 associated with Shc, Grb2 associated with Shc, and phosphorylated p44 MAPK was quantitated by densitometry. The intensity of the band derived from insulin-treated WT-hSHIP2-expressing cells was assigned a value of 1 arbitrary unit, and intensity of all treated groups was expressed as a fold value of control. Results are represented by mean ± SE of three separate experiments. *, P < 0.05 vs. WT-hSHIP2 by one-way ANOVA with Scheffé test.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Akt is one of target molecules of PI3-kinase important for the metabolic actions of insulin (8, 36). Although Akt1 and Akt2 are the ubiquitously expressed predominant isoforms, their physiological roles appear to differ (8, 36). Mice with lacking Akt1 showed a defect in growth, whereas they are normal with respect to glucose tolerance (29). In contrast, mice lacking Akt2 demonstrated insulin resistance in target tissues of insulin (30). Knockdown of endogenous Akt2, but not Akt1, by RNAi-based gene silencing apparently inhibited insulin-induced glucose uptake in 3T3-L1 adipocytes (37). Thus, Akt2 appears to be more closely associated with the metabolic effects of insulin (29, 30, 37). We recently demonstrated that SHIP2 predominantly regulated insulin-induced phosphorylation of Akt2, but not Akt1, in 3T3-L1 adipocytes (38). SNP3 (L632I) was the mutation found to be more frequent in control subjects than in type 2 diabetic patients. Because the 5'-phosphatase activity of SHIP2 was decreased by the substitution of SNP3-hSHIP2, it is important to clarify whether the mutation has an impact on the phosphorylation of Akt. Interestingly, expression of SNP3-hSHIP2 inhibited insulin-induced phosphorylation of Akt2 less potently than expression of WT-hSHIP2. The inhibition of Akt2 phosphorylation appeared to be paralleled with PI(3,4,5)P₃ levels in the cells expressing SNP3-hSHIP2. Taken together, possessing the SNP3 substitution might be, at least in part, related to the protection from insulin resistance in Japanese subjects. In this regard, insulin resistance index assessed by homeostasis model assessment (HOMA-R) tended to be lower in type 2 diabetic subjects with SNP3-hSHIP2 compared with the patients without the mutant, although the difference was not statistically significant.

After insulin stimulation, SHIP2 is tyrosine-phosphorylated by which SHIP2 is able to interact with the PTB domain of tyrosine-phosphorylated Shc (11). We previously reported that expression of a mutant Y/F-SHIP2 in the C-terminal tyrosine phosphorylation site diminished insulin-induced tyrosine phosphorylation of SHIP2, resulting in the decreased association of SHIP2 with Shc (31). SNP5 (N982S)-hSHIP2 was a rare mutant found in one diabetic patient, and it was not statistically associated with the type 2 diabetes. However, because the SNP5 substitution was located adjacent to the PTB domain binding consensus motif (NPAY⁹⁸⁶), one can speculate that the substitution might affect tyrosine phosphorylation of SHIP2. Along this line, insulin-induced tyrosine phosphorylation of SHIP2 and subsequent association with Shc were decreased in SNP5-hSHIP2-expressing cells compared with WT-hSHIP2-expressing cells. Moreover, consistent with the previous report showing that SHIP2 binding to Shc competes with the association of Shc with Grb2 (11), insulin-induced Shc/Grb2 association and MAPK activation were increased in SNP5-hSHIP2-expressing cells compared with WT-hSHIP2-expressing cells. Because p44/42 MAPK activity is strongly implicated in the mitogenic aspect of insulin leading to the atherosclerosis (39), it is possible that the diabetic patient with SNP5-hSHIP2 is related to the development and progression of atherosclerosis. Our results are consistent with the recent report showing that a significant association of variants in SHIP2 with hypertension is found in British type 2 diabetes (21). Indeed, the patient with SNP5-hSHIP2 is hypertensive, whereas the apparent atherosclerotic changes are not currently seen based on the analysis of intima-media thickness of carotid artery and electrocardiogram, possibly because of short durations after the diagnosis of type 2 diabetes. Apparently, the precise clinical follow-up concerning the progression of atherosclerosis will be needed in the patient with SNP5-hSHIP2.

In addition to the possible involvement of SNP5-hSHIP2 in the mitogenic aspect, the degree of the inhibition of insulin-induced phosphorylation of Akt2 was less in SNP5-hSHIP2-expressing cells compared with WT-hSHIP2-expressing cells. This may also be due to the decreased tyrosine phosphorylation of SHIP2 with SNP5. After insulin stimulation, SHIP2 is known to be translocated from the cytosol to the plasma membrane; thereby, PI(3,4,5)P₃ is adequately hydrolyzed to PI(3,4)-bisphosphate (38). Shc association with SHIP2 via its tyrosine phosphorylation appears to be required for the adequate localization of SHIP2 leading to the regulation of Akt (31, 38). Thus, it is possible to speculate that the defective tyrosine phosphorylation of SHIP2 with decreased binding to Shc may affect the cellular localization of SNP5-hSHIP2, resulting in failure to effectively inhibit the insulin-induced phosphorylation of Akt2 in the SNP5-hSHIP2. As regards with SNP6, the substitution is located in the C-terminal proline-rich region of SHIP2. Although the role of this region in insulin signaling is unknown, the SNP6 substitution may affect the molecular association for the appropriate localization of SHIP2 functioning. Alternatively, SNP6 may directly affect the 5'-phosphatase activity possibly by changing the tertiary structure of SHIP2. More precise studies will be needed to clarify these issues.

In summary, we identified 10 SNPs, including a haplotype (SNPs 1–3) and four missense mutations (SNPs 3–6), in human SHIP2 genomic DNA with 106 type 2 diabetic patients and 100 control subjects in a Japanese population. Because the haplotype was more frequent in control subjects than in type 2 diabetic patients and because the 5'-phosphatase activity of SNP3-hSHIP2 (L632I) appears to be decreased, possessing the SNP3-hSHIP2 might protect from insulin resistance, at least in part, in the Japanese population. Furthermore, one type 2 diabetic patient carrying SNP5-hSHIP2 was found as a rare case. Because insulin-induced Shc/Grb2 association leading to MAPK activation was increased in SNP5-hSHIP2-expressing cells, the substitution may be related to the progression of atherosclerosis. Taken together, the mutation of SHIP2 appears to be implicated in the pathogenesis of insulin resistance and/or atherosclerosis in a Japanese population. Replication studies with a large number of subjects in independent cohort will be necessary to further evaluate the contribution of identified SNPs to the pathogenesis of insulin resistance and type 2 diabetes. In addition, clarification of the possible mutants in the promoter and regulatory regions of SHIP2 would also be important for further understanding the impact of SHIP2 expression on the pathological state.

	Acknowledgments

 
We thank Drs. Akira Sato, Katsuya Yamazaki, Minoru Iwata, Yukio Kawagishi, Masaharu Urakaze, and Manabu Ishiki (Toyama Medical and Pharmaceutical University, Toyama, Japan) for their assistance in enrollment of subjects; Drs. Kazue Takagi, Naoko Hirota, and Satoshi Moriya (Toyama Medical and Pharmaceutical University, Toyama, Japan) for their assistance of the genetic analyses; Drs. Kiyokazu Nemoto (Mitsui Knowledge Industry Co., Ltd., Tokyo, Japan) and Hyogo Horiguchi (Jichi Medical School, Tochigi, Japan) for their assistance in the statistical analyses; and Dr. Wataru Ogawa (Kobe University, Kobe, Japan) for kindly providing CHO-IR cells.

	Footnotes

 
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

First Published Online February 1, 2005

Abbreviations: CI, Confidence interval; IVS, intronic variant sequence; LD, linkage disequilibrium; PI, phosphatidylinositol; PTB, phosphotyrosine binding; SHIP2, Src homology 2-containing 5'-inositol phosphatase 2; SNP, single nucleotide polymorphism; TBS, Tris-buffered saline; UTR, untranslated region; WT, wild type.

Received August 30, 2004.

Accepted January 25, 2005.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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