Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite, Caloric Intake and Fat Storage in Drosophila

Abstract

Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C14:0) and ceramide diene (C14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.

Ceramide-Protein Interactions Modulate Ceramide-Associated Lipotoxic Cardiomyopathy

“…Compared with the 4414 -Gal4 or UAS- Cdase -RNAi control flies, Cdase, Sk1 , and Sk2 KD flies exhibited wider diastolic and systolic diameters (Figures 1D, 1E, S1B, and S1C), resulting in reduced fractional shortening (Figures 1F and S1D) and contractile defects such as non-contractile regions and asynchronous beating (Figures 1G and S1E) (Diop et al, 2015). Of note, because global Sk1 and Sk2 KD also induced fat accumulation (Figure S1B) (Walls et al, 2013), these data suggest that these sphingosine kinases are required systemically for optimal overall lipid homeostasis and heart function.…”

“…We previously showed that knockdown (KD) of sphingosine kinase 2 ( Sk2 ) in Drosophila promoted ceramide and lipid/TAG accumulation through dysregulation of fat storage, lipogenic gene programs, and caloric uptake (Walls et al, 2013). Using the GAL-4/UAS system, we found that global KD of two additional other genes in the ceramide degradative pathway, Cdase (ceramidase) and Sk1 (sphingosine kinase 1), also caused elevated levels of ceramide (amide bond linked fatty acid: C 24 , C 22 , and C 20 ) and TAG (Figures 1B and S1A) compared with levels in control flies ( w 1118 , 4414 -Gal4).…”

“…We next investigated the consequences of whole-organism genetic inhibition of ceramide degradation—causing ceramide accumulation (Walls et al, 2013)—on heart function in 3-weekold females by using SOHA, a semi-intact specimen preparation combined with high-speed video microscopy (Fink et al, 2009; Ocorr et al, 2009). Representative M-mode traces are shown in Figure 1C.…”

“…In Drosophila , lace encodes SPT, which catalyzes the rate limiting step of de novo ceramide synthesis (Adachi-Yamada et al, 1999), while schlank encodes ceramide synthase, which catalyzes the addition of a second fatty acid chain to sphingosine to form ceramide (Bauer et al, 2009) (Figure 1A). Previous work showed that global KD of lace induces a lean phenotype and reduces both ceramide and TAG levels, primarily due to increased TAG utilization and deactivation of a lipogenic gene program (Walls et al, 2013). A similar pheno-type (reduction in ceramide and TAG levels) is observed in schlank -deficient flies, where the reduction in lipids was partially attributed to suppression of SREBP processing, which is a major lipogenic regulatory pathway (Bauer et al, 2009).…”

Dihydroceramides: From Bit Players to Lead Actors

“…Pharmacological inhibition of DES1 recapitulated the anti-adipogenic effects in vitro and in vivo. By contrast, the genetic ablation of the Des1 homolog in flies promoted fat storage concomitantly with increased level of dihydroceramides (78).…”