Kazuya Kabayama

Mice lacking ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function.

Glycosphingolipid deficiency affects functional microdomain formation in Lewis lung carcinoma cells.

In view of the increasing evidence that gangliosides in membrane microdomains or rafts are closely associated with various signal transducing molecules including Src family kinases, we compared rafts in two subclones of 3LL mouse lung carcinoma cell line, J18 and J5, characterized by high and very low GM3 ganglioside contents, respectively. Rafts were isolated from cell lysates as low density detergent-insoluble microdomains (DIM) by sucrose density gradient centrifugation. J5 and J18 cells expressed comparable amounts of Src family kinases and the majority of Src kinases in both clones were concentrated in their DIMs, suggesting that GM3 is not necessary for DIM localization of Src kinases and there is no direct interaction between Src and GM3. However, the Src kinases were eliminated from DIMs after depletion of the major neutral GSLs of J5 cells, glucosylceramide and lactosylceramide, by an inhibitor of glucosylceramide synthase (D-PDMP), indicating that GSLs in general are required for Src kinase association to DIM. J5 and the D-PDMP-treated J5 cells had very similar DIM protein profiles and moreover cholesterol and sphingomyelin in the GSL-depleted cells were enriched in DIM similar to the untreated control cells. Interestingly, the levels of tyrosine-phosphorylated DIM proteins and cell proliferation of J5 cells were much lower than those of J18 cells, suggesting that GM3 might be involved in tyrosine phosphorylation of DIM proteins required for cell growth. Thus, our data suggest that GSLs are essential for functional raft formation.

GM3 synthase gene is a novel biomarker for histological classification and drug sensitivity against epidermal growth factor receptor tyrosine kinase inhibitors in non‐small cell lung cancer

Expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell cycle phases, brain development and cancer malignancy. To investigate the characteristics of GM3 synthase, SAT‐I mRNA and ganglioside GM3 expression levels in lung cancer, we examined the expression levels of SAT‐I mRNA as well as GM3 in 40 tumor tissues surgically removed from non‐small cell lung cancer patients. Adenocarcinoma tissues expressed SAT‐I mRNA levels that were significantly higher than those of squamous and other carcinomas (P < 0.0001). Moreover, the SAT‐I mRNA levels were high in the bronchioalveolar carcinoma subtype and low in the solid and mucin subtypes of adenocarcinomas (P = 0.049, 0.049 and 0.013, respectively). To clarify the relationship between SAT‐I mRNA and epidermal growth factor receptor (EGFR)‐tyrosine kinase (TK) inhibitor sensitivity, we carried out drug sensitivity tests for the EGFR‐TK inhibitors gefitinib and AG1478 using eight adenocarcinoma cell lines expressing no EGFR mutations. The IC50 values for gefitinib and AG1478 decreased dramatically with increasing SAT‐I mRNA levels (R2 = 0.81 and 0.59, respectively), representing a wide range of drug sensitivities among adenocarcinoma cell lines. To explore a possible mechanism of how GM3 could enhance the sensitivity to EGFR‐TK inhibitors, the SAT‐I gene was introduced stably into a GM3‐negative clone of murine 3LL lung cancer cells to produce GM3‐reconstituted clones. We found an increase in EGFR protein levels and gefitinib sensitivity in GM3‐reconstituted cells, suggesting the involvement of GM3 in the turnover of EGFR protein. Therefore, it is highly expected that, by measuring the expression levels of SAT‐I mRNA in lung biopsy samples from non‐small cell lung cancer patients, enhanced pathological identification and individualized chemotherapeutic strategies can be established for the appropriate use of EGFR‐TK inhibitors. (Cancer Sci 2007; 98: 1625–1632)

Triglyceride accumulation and altered composition of triglyceride-associated fatty acids in the skin of tenascin-X-deficient mice.

Tenascin‐X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX‐deficient mice in comparison with that of wild‐type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX‐deficient (TNX−/−) mice. By thin‐layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX−/− mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX−/− mice. By gas chromatography‐mass spectrometry analysis of triglyceride‐associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX−/− mice compared with those in wild‐type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock‐transfected cells were also caused by over‐expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride‐associated fatty acids.

Glycosphingolipids govern gene expression.

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of β1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGFα receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven. Published in 2004.

Possible association of Neu2 with plasma membrane fraction from mouse thymus exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5

Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4MU‐Neu5Ac as a substrate. The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP‐40 or DOC/NP‐40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same pI as that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris‐buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane‐rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2‐transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.

Insulin Resistance and Type 2 Diabetes as Microdomain Disease: Implication of Ganglioside GM3

Caveolae are a subset of membrane microdomains (lipid raft) particularly abundant in adipocytes. Critical dependence of the insulin metabolic signal transduction on caveolae/microdomains in adipocytes has been demonstrated. These microdomains can be biochemically isolated with their detergent insolubility and were designated as detergent resistant microdomains (DRM). Gangliosides are known as structurally and functionally important components in microdomains. We demonstrated that increased GM3 expression was accompanied in the state of insulin resistance in mouse 3T3-L1 adipocytes induced by TNFα and in the adipose tissues of obese/diabetic rodent models such as Zucker fa/fa rats and ob/ob mice (Tagami et al. 2002). We examined the effect of TNFα on the composition and function of DRM in adipocytes and demonstrated that increased GM3 levels result in the elimination of insulin receptor (IR) from the DRM while caveolin and flotillin remain in the DRM, leading to the inhibition of insulin’s metabolic signaling (Kabayama et al. 2005). These findings are further supported by the report that mice lacking GM3 synthase exhibit enhanced insulin signaling (Yamashita et al. 2003). Thus, we present a new pathological feature of insulin resistance in adipocytes induced by TNFα.

A New Pathological Feature of Insulin Resistance and Type 2 Diabetes: Involvement of Ganglioside GM3 and Membrane Microdomains

Membrane microdomains (lipid rafts), which are critical for proper compartmentalization of insulin signaling, also play a role in the pathogenesis of insulin resistance, and yet this role has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in low density fractions, are rich in cholesterol, glycosphingolipids and various signaling molecules. TNFφ induces insulin resistance in type 2 diabetes, but its action mechanism is not fully understood. We found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFφ, suggesting a specific function for GM3. We extended these in vitro observations to living animals using obese Zucker fa/fa rats and ob/ob mice, in which the GM3 synthase mRNA levels in the white adipose tissues are significantly higher than in their lean controls. In DRMs from TNFφ-treated 3T3-L1 adipocytes, GM3 levels were doubled those of normal adipocytes. Additionally, insulin receptor (IR) accumulations in DRMs were diminished, while caveolin and flotillin levels were unchanged. GM3 depletion counteracted the TNFφ-induced inhibition of IR accumulation into DRMs. Together, these findings provide compelling evidence an insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3. Therefore, it is likely that life-style related diseases, such as type 2 diabetes, are membrane microdomain disorders caused by aberrant expression of glycosphingolipids.