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. 2011 Jul;52(7):1392-9.
doi: 10.1194/jlr.M014266. Epub 2011 May 5.

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Ayako Kohyama-Koganeya  1 , Takuji Nabetani, Masayuki Miura, Yoshio Hirabayashi
Affiliations

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Ayako Kohyama-Koganeya et al. J Lipid Res. 2011 Jul.

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
Altered dGlcT-1 expression in the fat body results in impaired energy homeostasis. A: Staining of lipid droplets that store TAG in the fat bodies of adult flies. Scale bar, 50 μm. Genotypes were as follows: control (FB-Gal4/+; +/+), UAS-dGlcT-1 (FB-Gal4/+; UAS-dGlcT-1/+), UAS-dGlcT-1 IR (FB-Gal4/+; UAS-dGlcT-1 IR/+). B: Drosophila GlcT-1 (dGlcT-1) knockdown clones (GFP-positive) harbored fewer and smaller lipid droplets (red) than controls (GFP negative). The Flp/FRT system was used to make dGlcT-1 knockdown clones in the fat body. dGlcT-1 knockdown clones were marked with GFP (hs-flp, UAS-GFP; tub-Gal4/UAS-dGlcT-1 IR; FRT82B Gal80/FRT82B). Scale bar, 50 μm. C: Measurement of whole-body TAG levels of 6 day-old male flies; n = 5 for each genotype. D, E, F: Stored carbohydrate levels were measured: glucose (D), trehalose (E), and glycogen (F); n = 8 for each genotype. The error bars represent SD.
Fig. 2.
Fig. 2.
ACC, FAS, and PEPCK mRNA expression is altered in dGlcT-1 knockdown fat bodies. A: Schematic diagram of the pathway and critical enzymes for TAG and glycerol biosynthesis. B: qRT-PCR was used to measure ACC, FAS, and PEPCK mRNAs involved in TAG and glycerol biosynthesis. FB-Gal4 was used to express UAS-dGlcT-1 IR specifically in third instar larval fat body; n = 3 for each genotype. C: Western blot analysis using anti-ACC indicated that changes in ACC mRNA expression are in good agreement with the levels of ACC enzyme protein. Proteins were collected from third instar larval fat body. The error bars represent SD.
Fig. 3.
Fig. 3.
Genetic interaction between dGlcT-1 and the p38-ATF2 signaling pathway. A: Schematic of the p38-ATF2 signaling pathway. B: Lsp2-Gal4 was used to overexpress or suppress dGlcT-1 specifically in the fat body of larvae, and the level of dp38 phosphorylation was analyzed by Western blotting with phospho-p38 antibody. C–E: Effect of dGlcT-1 overexpression or knockdown on PEPCK mRNA (C) and on stored lipid levels (D, E) in dATF2-overexpressing fat bodies of larvae. As described above, Lsp2-Gal4 was used to express dATF2 specifically in the fat body. In C, PEPCK mRNA levels were measured by qRT-PCR; n = 3 for each genotype. In E, whole-body TAG levels of third (L3) instar larvae were measured; n = 5 for each genotype. The error bars represent SD.
Fig. 4.
Fig. 4.
GlcCer is the sole GSL in the fat body. A: TLC analysis of GSLs from the fat body demonstrated that GlcCer is the sole GSL in the fat body. Total lipid extracts corresponding to two larvae were assayed (FB). Two micrograms of GlcCer, LacCer, and ganglioside GM3 were applied as standard GSLs (STD). B: LC/MS spectra from control (upper panel) and dGlcT-1-overexpressing fat bodies (FB>dGlcT-1; lower panel). Scans from m/z 690 to 710 are shown. Peaks of m/z 698.6 and 700.6 correspond to GlcCer having d14:1/20:0 and d14:0/20:0 conformations, respectively. C: Total GlcCer and MacCer levels in lipid extracts from fat bodies of control, FB>dGlcT-1, and FB>dGlcT-1 IR larvae are shown. D: Model showing the effect of overexpressing dGlcT-1 in the fat body. When dGlcT-1 is overexpressed in the fat body, dGlcT-1 adds glucose not only to regular ceramide but also to dihydroceramide (dotted line).
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