Takuji Nabetani

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs). Obesity is a metabolic disorder caused by an imbalance between energy uptake and expenditure, resulting in excess stored body fat. Recent studies have shown that GSL levels are increased in obese rodents and that pharmacologically reducing GSL levels by inhibiting GlcCer synthesis improves adipocyte function. However, the molecular mechanism underlying these processes is still not clearly understood. Using Drosophila as a model animal, we report that GlcT-1 expression in the fat body, which is equivalent to mammalian adipose tissue, regulates energy metabolism. Overexpression of GlcT-1 increases stored nutrition (triacylglycerol and carbohydrate) levels. Conversely, reduced expression of GlcT-1 in the fat body causes a reduction of fat storage. This regulation occurs, at least in part, through the activation of p38-ATF2 signaling. Furthermore, we found that GlcCer is the sole GSL of the fat body, indicating that regulation of GlcCer synthesis by GlcT-1 in the fat body is responsible for regulating energy homeostasis. Both GlcT-1 and p38-ATF2 signaling are evolutionarily conserved, leading us to propose an evolutionary perspective in which GlcT-1 appears to be one of the key factors that control fat metabolism.

Improved method of phosphopeptides enrichment using biphasic phosphate‐binding tag/C18 tip for versatile analysis of phosphorylation dynamics

A number of factors including low stoichiometry of phosphorylation, ion suppression, and reduced peptide backbone fragmentation interfere with precise identification of proteins in phosphoproteomic analysis by MS. Therefore, enrichment of phosphopeptides is an important process for subsequent mass spectrometric analysis. Here, we have developed a simple and efficient method for phosphopeptides enrichment, which employs a biphasic phosphate‐binding tag (Phos‐tag)/C18 tip consisting of overlaid Phos‐tag on the C18 resin in a pipet tip. The improvement in selectivity for phosphopeptides was achieved by using a 40% ACN solution under the phosphopeptides binding conditions. We also assessed the adequacy of Phos‐tag/C18 tip for quantitative phosphoproteomic analysis using the iTRAQ technology. After protein digestion and subsequent iTRAQ labeling, interfering substances including excess iTRAQ reagent were directly removed by Phos‐tag/C18 tip in a single step. Applying this method, phosphoproteomic analysis of HeLa cells stimulated with tumor necrosis factor ‐α was rapidly and successfully achieved.