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.

Effect of honeycomb-patterned surface topography on the function of mesenteric adipocytes.

Excessive accumulation of visceral adipose tissue, particularly mesenteric adipose tissue, is one of the most important factors in the pathogenesis of the metabolic syndrome. We previously developed a system for physiologically differentiating rat mesenteric-stromal vascular cells (mSVCs) to mesenteric-visceral adipocytes (mVACs) and are currently implementing various approaches to elucidate the details of the pathophysiology of mVACs. However, there is a critical problem to overcome, namely, that mature mVACs detach from the culture dishes and lose their function after approx. 10 days in culture. Therefore, we examined a culture of mSVCs on self-organized honeycomb-patterned films (honeycomb films) in order to establish a long-term culture for mVACs. The honeycomb films with highly regular porous structures can be prepared under humid casting conditions. These films can be prepared with ease, at a low cost and without any limitations pertaining to the availability of materials for the scaffold. As a result, mSVCs differentiated to mVACs and maintained their function for the secretion of adiponectin on the honeycomb films for at least 40 days. In addition, we investigated the influence of the pore size of the honeycomb films on mVAC behavior. We found that a honeycomb film with a pore size of 20 µm showed the highest mVAC function and optimum size for the long-term culture of mVACs. Thus, we established a long-term culture system for mVACs using the honeycomb films. We believe that this culture system will contribute to the understanding of the pathophysiology of mVACs and to the evaluation of drug candidates for the metabolic syndrome.

Physiological levels of insulin and IGF-1 synergistically enhance the differentiation of mesenteric adipocytes.

Visceral adipose tissue, particularly mesenteric adipose tissue, is important in the pathogenesis of metabolic syndrome. Here, we present a physiologically relevant differentiation system of rat mesenteric-stromal vascular cells (mSVC) to mesenteric-visceral adipocytes (mVAC). We optimized the insulin concentration at levels comparable to those in vivo ( approximately 0.85 ng/ml) by including physiological concentrations of IGF-1. We found that the insulin-like growth factor (IGF-1) and insulin worked synergistically, since IGF-1 alone could induce CCAAT/enhancer binding protein alpha (C/EBPalpha) and adipocyte lipid binding protein (aP2) mRNA expression but not lipid droplet accumulation associated with maturation. Using real-time PCR analyses on 180 adipocyte-related genes, we identified a dramatic effect by IGF-1 plus insulin. We also demonstrated the state of insulin resistance at pathologically high insulin concentrations. This culture system will contribute to understanding the physiological differentiation process and the patho/physiology of mVAC.

Circulating levels of ganglioside GM3 in metabolic syndrome: A pilot study.

BACKGROUND: Insulin resistance is a characteristic feature of metabolic syndrome. Ganglioside GM3 [α-Neu5Ac-(2-3)-ß-Gal-(1-4)-ß-Glc-(1-1)-ceramide] may impair insulin sensitivity in adipose tissue. We investigated the relationship between serum GM3 levels and adiposity indices, as well as between serum GM3 levels and metabolic risk variables. METHODS: Study 1: we assessed serum GM3 levels in normal subjects and in patients with hyperglycemia and/or hyperlipidemia (HL). Study 2: we investigated the relationship between serum GM3 levels and metabolic risk variables in patients with type 2 diabetes. RESULTS: Study 1: serum GM3 levels were higher in hyperglycemic patients (1.4-fold), hyperlipidemic patients (1.4-fold) and hyperglycemic patients with hyperlipidemia (1.6-fold), than in normal subjects. Study 2: serum GM3 levels were significantly increased in type 2 diabetic patients with severe obesity (visceral fat area (VFA) >200 cm(2), BMI > 30). The GM3 level was positively correlated with LDL-c (0.403, p = 0.012) in type 2 diabetes mellitus, but not affected by blood pressure. In addition, the high levels of small dense LDL (>10 mg/dL) were associated with the elevation of GM3. CONCLUSIONS: Serum GM3 levels was affected by glucose and lipid metabolism abnormalities and by visceral obesity. GM3 may be a useful marker for severity of metabolic syndrome.

Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNFalpha-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR-Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR beta-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR-Cav1 complex by the interactions of IR with GM3 in microdomains.

Role for up-regulated ganglioside biosynthesis and association of Src family kinases with microdomains in retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

Mouse F9 embryonal carcinoma cells have been widely used as a model for studying the mechanism of embryonic differentiation, because they are similar to the inner cell mass of early mouse embryos and can differentiate into primitive endoderm (PrE) following retinoic acid (RA) treatment. During F9 cell differentiation, the carbohydrate chains of glycoproteins and their corresponding glycosyltransferases are known to undergo rapid changes. However, there have been no corresponding reports on the expression of gangliosides. We have developed a custom cDNA array that is highly sensitive for the genes responsible for sphingolipid (SL) biosynthesis and metabolism. Using this, we found that, of the 28 selected genes, 26 exhibited increased expression during F9 differentiation into PrE. Although neutral glycosphingolipids (GSLs) were expressed at similar levels before and after differentiation, a greater than 20-fold increase in total ganglioside content was evident in PrE. Glucosylceramide synthase inhibitors (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol [d-PDMP] and its analog) depleted gangliosides and this resulted in delayed expression of Disabled-2 (Dab-2), suggesting the involvement of gangliosides in F9 cell differentiation. Disruption of cholesterol-enriched membrane microdomains by methyl-beta-cyclodextrin (MbetaCD) also delayed differentiation. Both MbetaCD and d-PDMP blocked the accumulation of Src family kinases (SFKs) to microdomains. However, d-PDMP did not block flotillin accumulation, yet MbetaCD did. Additionally, confocal laser microscopy revealed the formation of distinct functional microdomains integrating SFKs with gangliosides and cholesterol during PrE differentiation. Thus, we demonstrate the outstanding up-regulation of ganglioside biosynthesis and its importance in the formation of distinct microdomains incorporating SFKs with gangliosides during RA-induced differentiation of F9 cells.

TNFalpha-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3.

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. Tumor necrosis factor alpha (TNFalpha) induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. In other studies 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. In the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled compared with results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, whereas caveolin and flotillin levels were unchanged. Furthermore, insulin-dependent IR internalization and intracellular movement of the IR substrate 1(IRS-1) were both greatly suppressed in the treated cells, leading to an uncoupling of IR-IRS-1 signaling. GM3 depletion was able to counteract the TNFalpha-induced inhibitions of IR internalization and 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.

Comparison of the compositions of phospholipid-associated fatty acids in wild-type and extracellular matrix tenascin-X-deficient mice.

Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. We previously showed that TNX regulates the synthesis of triglyceride and the composition of triglyceride-associated fatty acids. The aim of the present study was to determine whether TNX controls the synthesis of phospholipids and the composition of phospholipid-associated fatty acids by using TNX-deficient (TNX-/-) mice and TNX-overexpressing fibroblast cell lines. Thin-layer chromatography of total lipids of the skin and sciatic nerves from wild-type and TNX-/- mice revealed that the amounts of major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in wild-type and TNX-/- mice are not different. Gas chromatography-mass spectrometry showed that the major fatty acid compositions of PC and PE in wild-type and TNX-/- mice are almost the same. Fibroblast cells stably overexpressing TNX also showed almost the same amounts of PC and PE and almost the same fatty acid compositions of PC and PE as those in mock-transfected cells. These results suggest that TNX regulates the amount of triglyceride and the composition of triglyceride-associated fatty acids but not the amount and species of phospholipids or the composition of phospholipid-associated fatty acids.

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.