Takashige Sato

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 TNFα-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 β-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.

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

SUMMARY BACKGROUND Insulin resistance is a characteristic feature of metabolic syndrome. Ganglioside GM3 [α-Neu5Ac-(2-3)-β-Gal-(1-4)-β-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.

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 (∼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/EBPα) 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.

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.

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.