Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice*

Akinori Sasaki {ddagger} § , Keiko Hata {ddagger} , Susumu Suzuki ||, Masashi Sawada {ddagger}, Tadashi Wada {ddagger}, Kazunori Yamaguchi {ddagger}, Masuo Obinata **, Hiroo Tateno {ddagger}{ddagger}, Hiroshi Suzuki § and Taeko Miyagi {ddagger} §§

From the Divisions of {ddagger}Biochemistry and {ddagger}{ddagger}Pathology, Research Institute, and the §Department of Internal Medicine, Miyagi Prefectural Cancer Center, Natori, Miyagi 981-1293, the ||Department of Molecular Metabolism and Diabetes, Tohoku University School of Medicine, Sendai 980-8574, and the **Department of Cell Biology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-8575, Japan

Received for publication, December 2, 2002 , and in revised form, April 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18–22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased {beta}-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides are a family of sialic acid-containing glycosphingolipids present in the cell surface membranes. Several lines of evidence suggest their important functional roles in regulating a wide range of biological processes including cell growth, cell differentiation, and transmembrane signaling (13). Most of the observations on ganglioside function, however, have been performed so far by using bacterial sialidases and exogenous gangliosides to mimic in vivo expression. To obtain further insights into their physiological significance and regulation mechanism, it is necessary to focus on endogenous sialidases responsible for ganglioside hydrolysis inside the cells. Three types of mammalian sialidase have been cloned and established to have low identity with each other in their primary structure. They differ in enzymatic properties and subcellular localization, being mainly found in lysosomes (Neu1), cytosol (Neu2), and plasma membranes (Neu3). Other than the contribution of lysosomal sialidase to glycoconjugate catabolism in lysosomes, their cellular roles are not well understood. The fact that plasma membrane sialidase is unique in specifically hydrolyzing gangliosides (410) and in its subcellular localization in plasma membranes, where levels of other glycosidases are very low, suggests participation in cell surface events through modulation of gangliosides. To obtain evidence of this, we previously cloned sialidase cDNAs of mammalian origin (8, 9, 11, 12), and we have recently investigated effects of overexpression in transgenic mice by using the human ortholog NEU3 cDNA. Unexpectedly, we have found that they develop insulin-resistant diabetes mellitus. To our knowledge, this is also the first example of glycosidase acting as a phosphorylated form and interacting with Grb2 via the SH2 domain.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Transgenic Mice—A 1.28-kb fragment containing the entire coding sequence of human NEU3 was inserted into the EcoRI site of the pCAGGS expression vector (13) under the {beta}-actin promoter (pCA-HmSD). A 3.5-kb fragment obtained by partial digestion of the plasmid with SalI and HindIII sites was inserted into Bluescript vector and then purified by means of CsCl2 gradient centrifugation. The SalI and HindIII fragment excised (Fig. 1a) was used for microinjection into the pronuclei of fertilized mouse eggs (DAB/2 x C57BL/6 F1). Integration of the transgene was detected by Southern blots of DNA extracted from tail biopsies using the 1.28-kb NEU3 fragment as a probe. Transgene positive mice (F0) were mated with C57BL/6 mice, and offspring (F1) from these crosses carrying transgenes were mated with each other. Subsequent generations were produced by further crosses of transgene-positive littermates. Two of these lines were chosen in this study. Age-matched transgene-negative littermates and their wild type were used as controls. Mice were fed an autoclaved formula diet and water ad libitum and handled in accordance with the Animal Care Committee of Miyagi Prefectural Cancer Center.



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FIG. 1.
Structure and expression of the NEU3 transgene. a, the NEU3 transgene used to generate the transgenic mice. The construct is for human NEU3 cDNA inserted into the pCAGGS vector. b, quantitative RT-PCR of total RNAs from various tissues of transgenic mice. The 0.8-kb band represents RT-PCR products specific to the exogenous NEU3 gene. c, NEU3 sialidase activity measured with gangliosides as substrate in the presence of Triton X-100.

 

Expression levels of the transgene were measured by quantitative RT-PCR analysis with a cDNA competitor prepared by NcoI digestion of Bluescript vectors containing entire open reading frames of NEU3. Primers were sense (5'-GACAGAGGGATTACCTACCGGATC-3', nucleotides 55–78 from the start codon) and antisense (5'-GAGCCATGATTCTGACGGTGTT-3', nucleotides 966–987). First strand cDNAs were synthesized from total RNA of the tissues by reverse transcription and then used as templates for PCR as described previously (14). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)1 was also measured as an internal control using a GAPDH competitor, prepared by digestion of the 0.5-kb cDNA (nucleotides 566–1017) with StyI and BspMI. After electrophoresis of amplified products, gel photos were subjected to densitometric analyses.

Cell Culture and Transfection—Mouse 3T3-L1 cells and rat L6 myoblasts (Health Science Research Resources Bank, JCRB 9014 and JCRB9081, respectively) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics. To differentiate 3T3-L1 cells into adipocytes, the cells were cultured for 2 days after reaching confluence in DMEM containing 10% FBS, 5 µg/ml insulin, 0.5 mM 3-isobutylmethylxanthine, 0.25 µM dexamethasone (15), followed by a 5-day culture in DMEM containing 10% FBS. The cells thus differentiated were transiently transfected by electroporation (8) with the NEU3 expression plasmid, pCA-HmSD, and after 48 h the cells were transferred to serum-starved conditions and stimulated with 100 ng/ml insulin for 10 min at 37 °C prior to harvesting. To evaluate the extent of differentiation, GAPDH activity was determined in the crude cell homogenates spectrophotometrically by measuring the oxidation of NADH at 340 nm using dihydroxyacetone phosphate as substrate (16). Rat L6 myoblasts were transiently transfected with pCA-HmSD using Effectene (Qiagen) reagent. After 36 h, the cells were transferred to serum-starved conditions, cultured for a further 16–20 h, and then stimulated with insulin as described above. The phosphorylation mutant (Y346A) was prepared by recombination PCR (17) with 5'-CTAGGTATCGCGCTCAACCAGACCCCCT-3' and 5'-CTGGTTGAGCGCGATACCTAGGTCAACC-3' primers and used for transfection in the same manner as a wild type pCA-HmSD.

Sialidase Activity Assays—Crude homogenates from tissues and cells were used for sialidase activity assays with mixed gangliosides (Sigma) as substrates in the presence of Triton X-100 as described previously (11, 12). One unit of sialidase was defined as the amount of enzyme catalyzing the release of 1 nmol of sialic acid/h.

Glucose and Insulin Tolerance Tests—After overnight fasting, mice were loaded with 1.75 g/kg glucose by intraperitoneal infusion. Blood was obtained from the tail vein, and blood glucose levels were measured before and 30, 60, and 120 min after glucose injection. Insulin levels were determined with an enzyme-linked immunosorbent assay insulin kit with mouse insulin as the standard (Morinaga, Tokyo, Japan). For the insulin tolerance test, mice were intraperitoneally challenged with 2 units/kg insulin after 5 h of fasting, and blood was taken as described above.

Histochemistry of Pancreatic Islets—Pancreatic tissue samples were fixed in 10% buffered formalin, pH 7.4, and stained with hematoxylineosin. Serial sections were stained immunohistochemically using polyclonal guinea pig anti-porcine insulin antibody (Zymed Laboratories Inc., San Francisco) with a DAB Substrate kit (Vector Laboratories, Burlingame, CA). Islet area and number were measured by computerized image analyzing techniques using NIH image. The areas of 23–25 islets per animal were calculated for five diabetic and five wild type mice.

Glycolipid Analysis by Thin Layer Chromatography—Glycolipids were extracted from mouse tissues as described elsewhere (18, 19) and fractionated by thin layer chromatography on high performance TLC plates (J. T. Baker Inc.) in chloroform/methanol/0.5% CaCl2 (60:40:9, v/v/v). Each lane of the plate was loaded with the equivalent of 6, 5, 100, 40, and 800 mg of wet weight of liver, brain, pancreas, kidney, and muscle, respectively. They were visualized with orcinol-H2SO4.

Analysis of Molecules Active in Insulin Signaling—To obtain the tissue specimens, mice fasted overnight were anesthetized with pentobarbital, and before and 5 or 15 min after insulin injection, hind limb muscle, liver, and epididymal adipose tissues were excised and immediately frozen in liquid nitrogen until analysis. Tissues and cells were homogenized and solubilized in 5 volumes of ice-cold buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2.5% deoxycholate, 2 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 2 mM phenylmethanesulfonyl fluoride, 7.5 µg/ml aprotinin, and 10 µg/ml leupeptin). The lysates, after centrifugation at 10,000 x g for 30 min, were used for immunoprecipitation of IR, IRS-1, Grb2, and NEU3 by incubation overnight at 4 °C with protein A/G-Sepharose and polyclonal antibodies (Santa Cruz Biotechnologies) to IR (C-19) and IRS-1 (C-20), as well as monoclonal antibodies to Grb2 (Transduction Laboratories) and NEU3, respectively. Monoclonal antibody (mAb) to human NEU3 was prepared as described previously (20). Western blotting was performed with lysates or immunoprecipitates separated on SDS-PAGE, and the proteins transferred to polyvinylidene difluoride membranes were immunoblotted with respective antibodies using the ECL Plus reagent (Amersham Biosciences). Tyrosine phosphorylation was detected with anti-phosphotyrosine-horseradish peroxidase conjugates (PY-20). To determine the effects of gangliosides on IR phosphorylation, IR was partially purified from skeletal muscle of C57BL/6 mice at 4 °C by a modification of the methods of Dubler et al. (21). Briefly, the particulate fractions of homogenates, prepared in 50 mM HEPES, pH 7.5, 8% sucrose, 10 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 1 µM pepstatin A, were solubilized with 2% Triton X-100 and further purified by sequential column chromatography on Sephacryl S-200 and wheat germ agglutinin-agarose. The IR fractions (1–3 µg of protein), with or without glycolipids, were incubated at room temperature for 50 min with or without 0.7 µM insulin. Phosphorylation was initiated with 250 µM ATP, 4 mM MgCl2, 5 mM MnCl2, and 0.1% Triton X-100, continued for 15 min at 4 °C, and terminated by addition of 0.4% Triton X-100, 20 mM EDTA, 200 mM KF, 20 mM Na3VO4, and 40 mM sodium phosphate, pH 7.0. The IR was then immunoprecipitated and analyzed as described above. PI 3-kinase activity was measured in immunoprecipitates obtained with antibody to IRS-1 as detailed earlier (22). Glycogen synthase activity was determined by a modification of the procedure of Thomas et al. (23), using incorporation of 0.5 mM UDP-[14C]glucose into glycogen at 30 °C for 15–30 min in the presence of glucose 6-phosphate (0.3 and 6 mM). Glutamine:fructose-6-phosphate amidotransferase activity was determined in the supernatant fraction from skeletal muscle as described previously (24). The mRNA level was assessed by RT-PCR using sense (5'-ATGTGTGGTATATTTGCTTACTT-3') and antisense (5'ATTCGGTGGATAGACAGACGGCC-3') primers based on the mouse cDNA (GenBankTM accession number U00932 [GenBank] ) (44).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of Diabetes in NEU3 Transgenic Mice—We generated NEU3 transgenic mice by using a human NEU3 cDNA (9) under the control of the {beta}-actin promoter (Fig. 1a). Human NEU3 was employed because of its strict ganglioside preference compared with the mouse counterpart. A total of six different transgenic mice, three males (TG1–3) and three females (TG4–6), were produced, containing 1–6 copies of the NEU3 gene as assessed by Southern blot hybridization (TG1 and TG4 contained 5 and 6 copies, respectively). The transgene was found to be expressed in a wide range of tissues, especially highly in muscle, pancreas, and heart, as shown by quantitative RT-PCR analysis (Fig. 1b). Sialidase activity assays with gangliosides as substrates revealed a 5–100-fold increase in transgenic as compared with wild type mice having the endogenous murine sialidase (Fig. 1c). The activity was not always parallel with the mRNA level, probably being affected by post-translational modifications. The transgenic mice grew normally and did not show any characteristic clinical signs by 15–17 weeks. To our surprise, however, the male TG1 and TG2 founders and the male offspring from four lines (TG1, -2, -4, and -5) developed impaired glucose tolerance with fasting hyperglycemia and hyper-insulinemia by 18–22 weeks. F3 through F5 offspring of TG1 and TG4 lines were used for the following experiments. Fasting blood glucose levels in the transgenic mice were significantly higher than in the wild type mice (187 ± 26 versus 98 ± 11 mg/dl). The blood glucose levels 120 min after intraperitoneal glucose load in the wild type and transgenic mice were 142 ± 12 and 491 ± 45 mg/dl, respectively, and fasting insulin levels were 0.23 ± 0.16 and 3.68 ± 0.62 µg/ml (Fig. 2a). Blood glucose level in the transgenic mice did not decrease so markedly as in the wild type mice even after injection of insulin (78.7 ± 12.4% versus 27.4 ± 0.9% of control after 120 min), as shown in Fig. 2b. To evaluate further this insulin resistance, homeostasis model assessment was determined according to the formula described by Matthews et al. (25). The values of 100 x (homeostasis model assessment-ratio) for the wild type and transgenic mice were 0.03 ± 0.03 and 2.37 ± 0.39 (p < 0.0001, n = 6), respectively, supporting extreme insulin resistance in the transgenic mice. These data indicate that the four lines of transgenic mice develop insulin-resistant diabetes mellitus in males, whereas the female founders and their offspring scarcely suffered from this abnormality. The average incidence of diabetes throughout eight generations was 75.4% in 62 male mice from the two founder lines at 21–25 weeks of age. The transgenic mice tended to be obese but did not show statistically significant difference in weight gain as compared with the wild type at 22 weeks of age: 34.63 ± 5.24 g for transgenic (n = 35) and 31.48 ± 7.57 g for wild type (n = 16) mice. Histological examination of pancreatic islets revealed remarkable hyperplasia in the diabetic mice (Fig. 2c), with statistically significant increase in area from 0.259 ± 0.050 mm2 (n = 5) to 0.587 ± 0.059 mm2 (n = 5) (p < 0.003) but no significant change in the islet number. A marked increase in {beta}-cell mass in islet was evident in the diabetic mice compared with the wild type mice, as immunologically positive insulin detected in enlarged islets of transgenic mice (Fig. 2c, lower panel), indicating overproduction of the hormone.



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FIG. 2.
Impaired glucose and insulin tolerance and hyperplasia of pancreatic islets in transgenic mice. a, glucose tolerance test. Wild type (n = 8) and transgenic mice (n = 8) were fasted 16 h before intraperitoneal injection with glucose (1.75 g/kg). Blood glucose levels (left) and insulin levels (right) of 18–22-week-old mice are determined. Data are mean ± S.D. values. Significant difference from the wild type value. (*, p < 0.01; **, p < 0.03.) b, insulin tolerance test. The mice were intraperitoneally challenged with 2 units/kg insulin after 5 h of fasting, and blood glucose levels were determined (*, p < 0.04; **, p < 0.02.) c, histochemistry of pancreatic islets. Hematoxylin-eosin staining (upper) and immunostaining for insulin (lower). x400.

 

Impaired Insulin Signaling in NEU3-transgenic Mice—To determine the molecular background underlying hyperglycemia and hyperinsulinemia in vivo, we examined tyrosine phosphorylation of IR and IRS-1 by immunoprecipitation with respective antibodies followed by immunoblotting with anti-phosphotyrosine antibody. In skeletal muscle from 24-week-old diabetic mice, insulin was less effective in stimulating tyrosine phosphorylation, with reduction by 51.5 ± 12.7% (n = 5) and 34.5 ± 10.5% (n = 5) for IR and IRS-1, respectively, 15 min after insulin injection (Fig. 3, a and b).



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FIG. 3.
Reduced insulin receptor signaling in transgenic muscle and in NEU3-transfected 3T3-L1 adipocytes. Insulin-stimulated phosphorylation of IR (a) and IRS-1 (b) in diabetic muscle (24-week-old mice). Equal amounts (0.5 mg) of solubilized muscle protein prepared from mouse strips were immunoprecipitated and immunoblotted with anti-phosphotyrosine (upper) and with anti-IR or IRS-1 (lower). The bar graphs on the right show data from five wild type and five transgenic (TG) subjects. Data are mean ± S.D. values. Significant difference from the wild type value (*, p < 0.02 for IR, and *, p < 0.01 for IRS-1). PI 3-kinase activity (c) and glycogen synthase activity (d) in diabetic muscle. Data are mean ± S.D. values for three subjects (*, p < 0.03 for PI 3-kinase, and p < 0.02 for glycogen synthase). 3T3-L1 adipocytes were transiently transfected with NEU3, and insulin-stimulated phosphorylation of IR and IRS-1 was estimated as shown in a representative blot (e).

 

By hypothesizing that PI 3-kinase and glycogen synthase, essential enzymes for regulation of glycogen synthesis in post-receptor insulin signaling, might be affected by the impaired insulin signaling, we compared their activity levels in skeletal muscle between diabetic mice and their normal counterparts. It can be seen from Fig. 3, c and d, that insulin stimulation of IRS-1-immunoprecipitable PI 3-kinase activity was low in diabetic mice compared with control muscle (3.5- versus 13.5-fold the basal level, respectively). Glycogen synthase activity in the diabetic subjects showed a tendency to be lower than in the control subjects under basal conditions, and reduction by 47.4% was evident after insulin stimulation. These results clearly indicate that insulin signaling is inhibited in the transgenic mice. Although activation of glutamine:fructose-6-phosphate amidotransferase, a key enzyme for hexosamine pathway, has been reported to lead to reduced sensitivity of glycogen synthesis to insulin (26), our results from measurement of the enzyme activity and mRNA levels in skeletal muscle did not show a significant increase but rather a slight decrease in the diabetic mice.

To obtain further evidence of reduced insulin response due to NEU3 overexpression, 3T3-L1 cells induced differentiation into adipocytes, and the NEU3 gene was then transiently introduced by electroporation into the cells. The transfectant showed 7.5-fold higher ganglioside sialidase activity than vehicle transfectants (22.10 ± 5.29 versus 2.95 ± 0.87 units/mg protein). When the extent of differentiation was examined by GAPDH assays, the transient transfectants showed GAPDH activity similar to vehicle transfectants (2.3 ± 0.6 as compared with 2.7 ± 0.5 units/mg for the control). Despite sufficient extent of differentiation, insulin-stimulated phosphorylation of IR and IRS-1 was significantly reduced in NEU3-transfected 3T3-L1 cells (Fig. 3e).

We then investigated which organs are primarily responsible for the insulin resistance and whether the defects occur in the younger mice before diabetic manifestation. In addition to muscle, liver, and adipose tissues from 24-week-old mice, the tissues from 8- to 9-week-old mice were used to assess IR phosphorylation (Fig. 4). As shown in muscle, a significant reduction was observed in liver and adipose tissues from the diabetic mice, and sensitivity to insulin was also attenuated in the tissues from the younger mice even without hyperglycemia, earlier in muscle than in liver and adipose tissues.



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FIG. 4.
IR phosphorylation in tissues from diabetic mice and in the younger subjects. Insulin-stimulated phosphorylation of IR was observed in muscle (a), liver (b), and adipose (c) tissues from diabetic mice (24-week-old) and younger mice before diabetic manifestation (8–9-week-old). TG, transgenic mice.

 

Regulation of IR Phosphorylation by Gangliosides—Next we investigated how NEU3 might be involved in the impaired insulin signaling. As gangliosides have been suggested to affect insulin receptor kinase activity (27, 28), possible alterations were examined by thin layer chromatography (Fig. 5a). GM1 and GM2 were increased as major products in various tissues from diabetic mice in comparison with control mice, which is reasonable because NEU3 was resistant to these gangliosides (9). In the case of muscle, there was decreased GM3 accompanied with somewhat increased lactosylceramide, although the total amount of gangliosides even in wild type muscle was less than a hundredth of that in liver. The possible effects of these and related glycolipids on insulin receptor action were examined in vitro using wheat germ agglutinin affinity-purified IR. As shown in Fig. 5, b and c, GM1 and GM2 inhibited IR phosphorylation by 45–70% at 45 µM, whereas GM3 and GD1a, good substrates for NEU3 sialidase, and lactosylceramide, another product of NEU3, exerted hardly any effects at this concentration. These data suggest that overexpressing NEU3 leads to down-regulated IR phosphorylation, probably at least partly due to accumulation of GM1 and GM2, because intracellular concentrations of these metabolites in several tissues including liver were comparable with those used in this in vitro experiment.



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FIG. 5.
Analysis of NEU3 reaction products by thin layer chromatography and their modulation of IR phosphorylation. a, thin layer chromatography of glycolipids from several tissues of wild type and transgenic mice. Stg, standard gangliosides (NeuGc); Sta, standard gangliosides (NeuAc); W, wild type; T, transgenic mice. b, reduction of IR phosphorylation by NEU3 reaction products. c, The graph on the right shows average values from three independent experiments.

 

Mechanism of Action of NEU3—To elucidate further the mechanism of action of NEU3, we investigated whether the protein is affected by insulin treatment. Because several phosphorylation sites exist in the primary structure, NEU3 protein was examined for phosphorylation in response to insulin stimulation in skeletal muscle by analyzing the state of protein immunoprecipitated with anti-NEU3 mAb. NEU3 was in fact tyrosine-phosphorylated, and the extent was increased in response to insulin (Fig. 6a), whereas no band was detectable in the case of wild type mice, as expected. To explore whether such phosphorylation influences the sialidase activity level, we measured the NEU3 activity with substrate gangliosides. As shown in Fig. 5a (right panel), the increased ratios by insulin were 2.8–3.6- and 3.0–4.2-fold for the activity and phosphorylation, respectively, in the transgenic muscle. In proportion to phosphorylation, higher sialidase activity was yielded, suggesting that the activity level of NEU3 may be regulated by tyrosine phosphorylation in skeletal muscle responding to insulin. Because we postulated that Grb2 adaptor protein, which contains SH2 and SH3 domains for interaction with signaling molecules (29, 30), might bind to the pYXNX motif (where pY is a phosphotyrosine) encompassing Tyr-346 of NEU3 via its SH2 domain, we examined this possibility using specific antibodies to Grb2 and NEU3 (Fig. 6b). With muscle extracts, NEU3 protein was co-immunoprecipitated with Grb2 by anti-Grb2 antibody, and Grb2 protein was detected in the immunoprecipitates with anti-NEU3 antibody, under conditions of almost equal amounts of endogenous Grb2. Larger amounts of NEU3 and Grb2 were recovered with Grb2 and NEU3, respectively, after insulin stimulation, indicating that tyrosine-phosphorylated NEU3 associates with Grb2 in response to insulin.



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FIG. 6.
Tyrosine phosphorylation, activation, and association with Grb2 of exogenous NEU3. a, tyrosine phosphorylation of NEU3 in muscle (left panel) and sialidase activity toward ganglioside substrates before and after insulin injection (right panel). b, co-immunoprecipitation of NEU3 in muscle with Grb2 in response to insulin. After immunoprecipitation with anti-Grb2 or -NEU3 mAb, NEU3 or Grb2 in the immunoprecipitates was detected with the respective antibodies. c, requirement of the tyrosine phosphorylation site of NEU3 for association with Grb2 and for negative regulation of IRS-1 phosphorylation. Cells transfected with vector alone, the Y346A mutant, or wild type were collected for immunoprecipitation with anti-Grb2 or -NEU3 mAb, and NEU3 protein in the immunocomplexes was detected with antiNEU3 mAb. IRS-1 phosphorylation was tested as detailed above.

 

To confirm whether the phosphorylation site of NEU3 is responsible for the binding to Grb2, we prepared a NEU3 mutant, Y346A, featuring substitution of Ala for Tyr-346, transfected into L6 myocytes (Fig. 6c). In response to insulin, the wild type NEU3 demonstrated a clearly enhanced association with Grb2, as observed in transgenic muscle. However, the mutant showed marked decrease in sialidase activity (5–7% of the wild type) and failed to co-immunoprecipitate with Grb2, although the NEU3 protein was as much expressed as in the wild type. Furthermore, IRS-1 phosphorylation scarcely changed in the mutant (the lowest panel in Fig. 6c), whereas the NEU3 wild type showed a significant reduction. Similarly, glycogen synthase activity was decreased in the wild type (67% of control), but the mutant showed the same level as control (data not shown). These data suggest that tyrosine phosphorylation and activation followed by association with Grb2 may be required for involvement of NEU3 in insulin signaling.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
This study provided the first evidence that sialidase is involved in regulation of insulin signaling and that unbalanced expression may lead to impaired insulin signaling and subsequently to insulin-resistant diabetes. Insensitivity to insulin already appeared before manifestation of hyperglycemia, suggesting that the observed defects are a result of NEU3 overexpression. The defects developed earlier in muscle than in liver and adipose tissues, and therefore muscle, which showed exogenous NEU3 activity most abundantly, is likely an organ primarily responsible for insulin resistance in the transgenic mice.

Previous observations with exogenous bacterial sialidases have indicated that sialidase-catalyzed loss of sialic acids from the insulin receptor protein (31) or some sialic acid-containing components close to the receptor (32, 33) could affect IR function. This concept does not conflict with our findings, although NEU3 cannot remove sialic acids from the receptor protein itself because of an inability to act on glycoproteins (410). It has been observed that endogenous sialidase activity in the kidney (34) and retina (35) of diabetic rats is higher than in controls, with 4-methylumbelliferyl-neuraminic acid as the substrate. However, this is not likely to be directly connected to the present results, because the substrate is targeted by lysosomal type sialidase, which could account for the observed activity. A recent report (36) describing increased membrane sialidase activity in erythrocytes of diabetic patients indicates that our findings are probably applicable to human diabetes. We are actually having possible evidence of polymorphism of the NEU3 gene in diabetic patients,2 and we are now analyzing how the genetic variation is associated with the clinical features of diabetic patients. At present we do not know the reason for the gender specificity for diabetes in our mice, but it is not a special case in diabetic animal models, as observed in Zucker diabetic fatty rats (37). The gender difference is perhaps due to gender-associated developmental restriction of expression of NEU3-related molecules or the contribution of putative genes in chromosome X.

Phosphorylation and subsequent activation of NEU3 in response to insulin may affect insulin signaling in two directions. One is the upstream pathway, where NEU3 down-regulates IR phosphorylation through modulation of gangliosides, and the other is in the downstream pathway, where it influences signaling through interaction with Grb2. With regard to the former, suppression of IR phosphorylation by accumulation of sialidase products would cause disturbance of downstream signaling and subsequent stimulation of insulin production to compensate, resulting in insulin-resistant diabetes. These effects are unlikely simply due to blockade of phosphorylation of endogenous substrates by overexpressed NEU3, because the mRNA level of exogenous NEU3 in the transgenic tissues was not so high, ~1/10 to 1/120 of {beta}-actin expression level (data not shown). Although gangliosides have been described as candidate autoantigens in type I diabetes (38), they probably act as signal modulators in our case. Under physiological conditions, insulin-stimulated IR should trigger phosphorylation of signaling-related molecules, including NEU3, and negative feedback control of IR phosphorylation by NEU3 products, at least partly, would take place to maintain a steady-state glucose level. There are several reports describing participation of GM3 as a negative regulator in insulin signaling (27, 28, 39, 40). It was shown that insulin resistance induced by tumor necrosis factor-{alpha} was accompanied by GM3 increase in 3T3-L1 adipocytes (28). In addition, mice lacking GM3 synthase were found to be protected from high fat diet-induced insulin resistance (40). From this point of view, NEU3 overexpression would be expected to lead to increased insulin sensitivity via accelerated degradation of GM3. The experimental conditions in these studies are different from one another, and therefore we cannot explain clearly why our results are inconsistent with those above. However, in our case, accumulated products such as GM1 and GM2 are likely to affect insulin sensitivity rather than a decrease in GM3, because concentrations of the former gangliosides employed for in vitro IR phosphorylation were roughly comparable with their intracellular concentrations in several tissues including liver. Furthermore, GM3 was hardly detectable even in the wild type liver and was detectable but extremely low (less than 1 µM) in the wild type muscle, whereas the ganglioside has an effect on IR phosphorylation only at much higher concentrations, as shown in Fig. 5b and in previous observations (27, 28) as well. It is still possible that highly specific but minute amounts of glycolipid products other than GM1 and GM2 cause the signaling defects in the transgenic mice.

As to downstream signaling via an interaction with Grb2, phosphorylated NEU3 may down-regulate this by recruitment of signal-related molecules through interaction with Grb2, as suggested by the data showing that NEU3 transfection into L6 cells caused reduction of IRS-1 phosphorylation and glycogen synthase activity. This possibility was strengthened by the results with a phosphorylation mutant, which failed to associate with Grb2 accompanied by loss of sialidase activity and recovered from impaired signaling. It is generally thought that association with Grb2 can affect Ras and mitogen-activated protein kinase signaling (29, 30) rather than glucose metabolism, but it is possible for an effect on post-receptor signaling, like the example of protein-tyrosine phosphatase 1B deactivating IRS-1 by complex formation with Grb2 (41). In this context, it is of interest that NEU3 is now considered to be located in the detergent-insoluble microdomains, lipid rafts (42), and caveolae (20), where recruitment of insulin signaling molecules occurs (43). In the two possible ways proposed here for NEU3 participation in insulin signaling, the former may have a greater influence on signaling than the latter, because it is based on a specific catalytic action of NEU3. Further studies are now needed to determine what molecules other than Grb2 are directly linked to the sialidase in insulin signaling, and whether and how alterations in the gene are involved in human diabetes. Our mice should provide a useful model for human diabetes and valuable tools for study of novel therapeutic approaches.


    FOOTNOTES
 
* This work was supported in part by grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, Culture and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Both authors contributed equally to this work. Back

§§ To whom correspondence should be addressed: Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, 47-1 Nodayama, Medeshima-shiode, Natori, Miyagi 981-1293, Japan. Tel.: 81-22-384-3151; Fax: 81-22-381-1195; E-mail: tmiyagi{at}mcc.pref.miyagi.jp.

1 The abbreviations used are: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; IR, insulin receptor; IRS-1, insulin receptor substrate; PI 3-kinase, phosphatidylinositol 3-kinase; mAb, monoclonal antibody; SH, Src homology; RT, reverse transcriptase. Back

2 Y. Hinokio, S. Suzuki, M. Hirai, Y. Oka, K. Yamaguchi, and T. Miyagi, manuscript in preparation. Back


    ACKNOWLEDGMENTS
 
We greatly appreciate the helpful advice of Dr. Yoshimi Homma for the PI 3-kinase activity assay (Institute of Biomedical Sciences, Fukushima Medical College), and we also thank Setsuko Moriya for expert technical assistance.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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