Perioperative changes in ganglioside monosialodihexosylganglioside (GM3) molecular species for benign prostatic hyperplasia: a preliminary report.

Benign prostatic hyperplasia (BPH) progresses with age and is associated with chronic inflammation. We focused on the relationship between BPH and ganglioside monosialodihexosylganglioside (GM3), a sialic acid-containing glycosphingolipid that is involved in chronic inflammation. GM3 molecular species would have a significant role in regulating inflammatory processes. In this prospective study, preoperative and postoperative serum samples were obtained from patients who underwent holmium laser enucleation of the prostate (HoLEP) for BPH. Preoperative and postoperative measurements of serum GM3 species were performed one month before and three months after HoLEP. Twenty-three patients were included in the study. The average patient age was 75 years, and the average prostate volume was 66 mL. The average weight of the surgically resected prostate tissue was 42 g. At three months after HoLEP, the serum concentration of GM3 species was found to have decreased after HoLEP compared with the preoperative concentration of GM3 species. Six GM3 species such as d18:1-17:0 [C17 acyl chain (-17:0) linked to a C18 sphingosine base with a double bond (d18:1-) by an amide linkage], were significantly reduced. The sample size was small; therefore, this study showed only preliminary results and could not evaluate prostate tissue inflammation. This study showed that the serum concentrations of several GM3 species, which indicate chronic inflammation, may be significantly reduced after BPH surgery.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

Roles of Gangliosides in Hypothalamic Control of Energy Balance: New Insights.

Gangliosides are essential components of cell membranes and are involved in a variety of physiological processes, including cell growth, differentiation, and receptor-mediated signal transduction. They regulate functions of proteins in membrane microdomains, notably receptor tyrosine kinases such as insulin receptor (InsR) and epidermal growth factor receptor (EGFR), through lateral association. Studies during the past two decades using knockout (KO) or pharmacologically inhibited cells, or KO mouse models for glucosylceramide synthase (GCS; Ugcg), GM3 synthase (GM3S; St3gal5), and GD3 synthase (GD3S; St8sia1) have revealed essential roles of gangliosides in hypothalamic control of energy balance. The a-series gangliosides GM1 and GD1a interact with leptin receptor (LepR) and promote LepR signaling through activation of the JAK2/STAT3 pathway. Studies of GM3S KO cells have shown that the extracellular signal-regulated kinase (ERK) pathway, downstream of the LepR signaling pathway, is also modulated by gangliosides. Recent studies have revealed crosstalk between the LepR signaling pathway and other receptor signaling pathways (e.g., InsR and EGFR pathways). Gangliosides thus have the ability to modulate the effects of leptin by regulating functions of such receptors, and by direct interaction with LepR to control signaling.

Serum GM3(d18:1-16:0) and GM3(d18:1-24:1) levels may be associated with lymphoma: An exploratory study with haematological diseases.

GM3 (monosialodihexosylganglioside) is a type of ganglioside, which is a molecule composed of ceramide and oligosaccharide containing one or more sialic acids. Since GM3 is abundantly expressed in blood cells, we investigated the association between GM3 molecular species and haematological diseases. We measured the serum levels of seven GM3 molecular species in subjects with various haematological diseases (n = 52) and healthy subjects (n = 24) using a liquid chromatography tandem-mass spectrometry technique as an exploratory study. In all the subjects with haematological diseases, GM3(d18:1-16:0) were inversely correlated with the erythrocytes counts. Regarding the difference in serum GM3 molecular species levels among each haematological diseases and healthy subjects, the levels of GM3(d18:1-16:0) and GM3(d18:1-24:1) were higher in the lymphoid neoplasm group than healthy subjects. Principal component analyses also revealed that the GM3(d18:1-16:0) and GM3(d18:1-24:1) levels were significant contributing factors for discriminating the lymphoid neoplasm group. Moreover, in the lymphoid neoplasm group, the GM3(d18:1-16:0) levels were significantly and positively correlated with the levels of C-reactive protein, soluble interleukin-2 receptor, and lactate dehydrogenase. In conclusion, in our exploratory study with haematological diseases, GM3 molecular species showed different distribution among disease groups, and serum GM3(d18:1-16:0) and GM3(d18:1-24:1) might be associated with lymphoma.

Overexpression of HexCer and LacCer containing 2-hydroxylated fatty acids in cholangiocarcinoma and the association of the increase of LacCer (d18:1-h23:0) with shorter survival of the patients.

Alteration of glycosphingolipid (GSL) synthesis is observed in many types of cancer. In this study, we have analyzed the expression of sphingolipids and GSLs in cholangiocarcinoma (CCA) tissues and adjacent normal liver tissues. Neutral lipids were extracted from tissue samples using mild-alkaline treatment method followed by TLC and LC-MS analysis. The expression of ceramides, hexosylceramides (HexCer), and lactosylceramides (LacCer) was altered in CCA tissues, 61.1% (11/18) of them showing an increase whereas 38.9% (7/18) showing a decrease, compared with the adjacent normal tissue. Cers and GSLs containing 2-hydroxylated fatty acids except one LacCer molecular species were overexpressed in CCA tissues, and the increase of LacCer (d18:1-h23:0) was correlated with shorter survival of CCA patients, suggesting the involvement of GSL synthesis and fatty acid hydroxylation in progression of CCA. Taken together, we have demonstrated in this study the increase of GSL synthesis and fatty hydroxylation in CCA, which probably be used as a target for CCA treatment.

Globo-series glycosphingolipids enhance Toll-like receptor 4-mediated inflammation and play a pathophysiological role in diabetic nephropathy.

Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inflammatory processes are involved in development and progression of diabetic nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles in inflammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role of these GSLs in this disease process. We studied proinflammatory activity of GSLs in diabetic nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD) (60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer), upregulation of TNF-α, and induction of renal inflammation. Gb3Cer/Gb4Cer treatment enhanced inflammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T, mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our findings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inflammatory response, and that such GSLs play an important pathophysiological role in diabetic nephropathy.

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment.

Nowadays we understand cell membranes not as a simple double lipid layer but as a collection of complex and dynamic protein-lipid structures and microdomains that serve as functional platforms for interacting signaling lipids and proteins. Membrane lipids and lipid structures participate directly as messengers or regulators of signal transduction. In addition, protein-lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains. Thus, lipid alterations change cell signaling that are associated with a variety of diseases including cancer, obesity, neurodegenerative disorders, cardiovascular pathologies, etc. This article reviews the newly emerging field of membrane lipid therapy which involves the pharmacological regulation of membrane lipid composition and structure for the treatment of diseases. Membrane lipid therapy proposes the use of new molecules specifically designed to modify membrane lipid structures and microdomains as pharmaceutical disease-modifying agents by reversing the malfunction or altering the expression of disease-specific protein or lipid signal cascades. Here, we provide an in-depth analysis of this emerging field, especially its molecular bases and its relevance to the development of innovative therapeutic approaches.

Control of homeostatic and pathogenic balance in adipose tissue by ganglioside GM3.

Ganglioside GM3 (Siaα2-3Galß1-4Glcß1-1Cer) has been known to participate in insulin signaling by regulating the association of the insulin receptor in caveolae microdomains (lipid rafts), which is essential for the execution of the complete insulin metabolic signaling in adipocytes. Macrophage-secreted factors including proinflammatory cytokines, tumor necrosis factor-α and interleukin-1ß, in adipose tissues have been known to limit the local adipogenesis and induce insulin resistance; however, the interplay between adipocytes and macrophages upon regulation of GM3 expression is not clear. GM3 was virtually absent in primary adipocytes differentiated from macrophage-depleted mesenteric stromal vesicular cells, which accompanies enhancement of insulin signaling and adipogenesis. We found that the expression of GM3 is governed by soluble factors including steady-state levels of proinflammatory cytokines secreted from resident macrophages. The direct involvement of GM3 in insulin signaling is demonstrated by the fact that embryonic fibroblasts obtained from GM3 synthase (GM3S)-deficient mice have increased insulin signaling, when compared with wild-type embryonic fibroblasts, which in turn leads to enhanced adipogeneis. In addition, GM3 expression in primary adipocytes is increased under proinflammatory conditions as well as in adipose tissue of diet-induced obese mice. Moreover, GM3S-deficient mice fed high-fat diets become obese but are resistant to the development of insulin resistance and chronic low-grade inflammatory states. Thus, GM3 functions as a physiological regulatory factor of the balance between homeostatic and pathological states in adipocytes by modulating insulin signaling in lipid rafts.

Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells.

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.

Impairment of hippocampal long-term potentiation and failure of learning in mice treated with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which has been suggested as the basal cellular process of learning and memory in the brain. In the present study, long-term potentiation (LTP) and long-term depression (LTD) in CA1 hippocampal neurons and learning behavior were examined in mice treated with (D)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ((D)-PDMP), an inhibitor of ganglioside biosynthesis. Mice treated with (D)-PDMP, but not those treated with (L)-PDMP, showed impairment of LTP induction in hippocampal CA1 neurons without any significant change in LTD formation and also showed a failure of learning in the 4-pellet taking test. These results indicate that de novo synthesis of gangliosides in the brain is involved in synaptic plasticity of LTP in mouse hippocampal CA1 neurons and plays important roles in learning and memory.

Dissociation of the insulin receptor from caveolae during TNFα-induced insulin resistance and its recovery by D-PDMP.

Previously, we demonstrated that an inhibitor of ganglioside biosynthesis, d-PDMP, could restore impaired insulin signaling in tumor necrosis factor α (TNFα)-treated adipocytes by blocking the increase of GM3 ganglioside. Here, we analyzed the interaction between insulin receptor (IR) and GM3 in the plasma membranes using immunoelectron microscopy. In normal adipocytes, most GM3 molecules localized at planar and non-caveolar regions. Approximately 19% of IR molecules were detected in caveolar regions. The relative ratio of IRs associated with caveolae in TNFα-treated adipocytes was decreased to one-fifth of that in normal adipocytes, but this decrease was restored by d-PDMP. Thus, we could obtain direct evidence that insulin resistance is a membrane microdomain disorder caused by aberrant expression of ganglioside.

The cytoplasmic tail of GM3 synthase defines its subcellular localization, stability, and in vivo activity.

GM3 synthase (SAT-I) is the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. In this study, we identify three isoforms of mouse SAT-I proteins, named M1-SAT-I, M2-SAT-I, and M3-SAT-I, which possess distinct lengths in their NH(2)-terminal cytoplasmic tails. These isoforms are produced by leaky scanning from mRNA variants of mSAT-Ia and mSAT-Ib. M2-SAT-I and M3-SAT-I were found to be localized in the Golgi apparatus, as expected, whereas M1-SAT-I was exclusively found in the endoplasmic reticulum (ER). Specific multiple arginines (R) arranged in an R-based motif, RRXXXXR necessary for ER targeting, were found in the cytoplasmic tail of M1-SAT-I, and in vivo GM3 biosynthesis by M1-SAT-I was very low because of restricted transport to the Golgi apparatus. In addition, M1-SAT-I and M3-SAT-I had a long half-life relative to M2-SAT-I. This is the first report demonstrating the presence of an ER-targeting R-based motif in the cytoplasmic tail of a protein in the mammalian glycosyltransferase family of enzymes. The system, which produces SAT-I isoforms having distinct characteristics, is likely to be of critical importance for the regulation of GM3 biosynthesis under various pathological and physiological conditions.

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 IC(50) values for gefitinib and AG1478 decreased dramatically with increasing SAT-I mRNA levels (R(2) = 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.

Insulin resistance as a membrane microdomain disorder.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. TNFalpha induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. We have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFalpha, suggesting a specific function for GM3. We were able to extend 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 the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled, compared to results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, while caveolin and flotillin levels were unchanged. GM3 depletion was able to counteract the TNFalpha-induced inhibition of IR accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.

Insulin resistance as a membrane microdomain disorder.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. TNFalpha induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. We have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFalpha, suggesting a specific function for GM3. We were able to extend 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 the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled, compared to results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, while caveolin and flotillin levels were unchanged. GM3 depletion was able to counteract the TNFalpha-induced inhibition of IR accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.