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

<div><p>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 <i>Drosophila</i> 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 (C_14:0) and ceramide diene (C_14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C_14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor <i>dNepYr</i>, resulting in caloric intake-dependent obesity.</p></div

Sphingolipidomic profiles of SL mutants.

<p><b>Sph = Sphingosine; S1P = Sphingosine 1-Phosphate; C = ceramide;</b> Dihydroceramide (C_14:0), ceramide (C_14:1) and ceramide diene (C_14:2) subspecies are shown and represent the degree of saturation on the sphingoid backbone. C_20:0, C_22:0 and C_24:0 denote the length and saturation of the second fatty acid chain connected to the sphingoid backbone in these ceramides.</p>*<p>denotes p-value<0.05.</p

Sphingolipid regulation of caloric intake and fat mobilization.

<p>Both saturated and unsaturated fats act as precursor “input” molecules in the production of “output” sphingolipid intermediates, specifically ceramide and S1P, which act to transduce a physiological response. (Left) Sphingolipid metabolism regulates Akh cell viability and function (Right). After a meal, S1P accumulates downstream of elevated ceramide. S1P, either directly or indirectly (through dRYamide), induces dNepY receptor signaling in the hindgut, inducing appetite suppression, reduced caloric intake and downregulation of <i>dNepYr</i> mRNA expression (negative feedback).</p

The ceramide:S1P rheostat's role in regulating <i>dNepYr</i> expression and caloric intake.

<p>(A) <i>dNepYr</i> expression is upregulated in <i>Sk2^KG6050894</i> and downregulated in <i>Sply⁰⁵⁹⁰¹</i> mutants, with concurrent changes in overlapping cis-NAT's of pancreatic TAG lipase genes. Two day old <i>Sk2^KG6050894</i> flies and wt flies were administered 0 uM, 1 uM, 10 uM and 100 uM dRYamide (1∶1 dRYamide 1∶2) in solid food for 6 days, after which (B) TG levels, (C) rate of dose dependent TG decline, and (D) <i>dNepYr</i> mRNA levels were measured. Three to five day old <i>Sk2^KG6050894</i> flies were also administered S1P analogue FTY720P in the CAFE, after which (E) caloric intake (F) and <i>dNepYr</i> mRNA levels were measured. Error bars are represented by the S.E.M. p-values<*0.05, **<0.01, ***p<0.001.</p

Differential expression of Lipid Metabolic and Apoptotic genes in SL mutants.

<p>DNA microarray analysis in conjunction with DAVID bioinformatics analysis was used to identify distinct subsets of genes mapped to elucidated pathways. Downregulation (Fold change <1.5) is shown in green while upregulation (Fold change >1.5) is shown in red, up to a maximum of 3-fold or greater difference. All changes >3 fold are represented by the brightest color. No change is displayed as black. (A)These data show that diene-accumulating, Akh cell-ablating <i>ifc⁴</i> mutants exhibit upregulation of proapoptotic genes and downregulation of anti-apoptotic genes, while diene-depleting, Akh cell-expanding <i>lace^k05305/2</i> mutants' exhibit downregulation of proapoptotic genes and upregulation of anti-apoptotic genes. (B) These data show that appetite suppressed S1P accumulating <i>Sply⁰⁵⁹⁰¹</i> mutants' downregulate lipogenic pathways (FA biosynthesis) and upregulate lipid utilizing pathways (Fatty acid Oxidation and Oxidative Phosphorylation). Conversely, high appetite, S1P depleting <i>Sk2^KG050894</i> mutants upregulate lipogenic pathways (specifically FA synthase) and downregulate lipid utilizing pathways.</p

Classic hallmarks of obesity.

<p>Obesity in flies is characterized by classic hallmarks of obesity observed in higher organisms. (A) Whole fly triglyceride (TAG) and (B) hemolymph TAG levels were measured in µg of TAG per mg flies and normalized to wt flies. (C) Resistance to starvation-induced death was measured as the mean % of the population that survives over time of 3 independent experiments. (n = 100 flies) (D) Mean body weight (mg) was measured in six sets of n = 100 flies. (E–K) One-Day old larval origin fat body cells stained with lipid positive Nile Red (Red) and a nuclear stain DAPI (blue). (L) Distribution of fat body cell size (µm) from 50 randomly selected fat bodies from n = 4 flies used to calculate (M) mean fat body cell size. Error bars are represented by the S.E.M. p-values *<0.05, **<0.01, ***<0.001.</p

SL Metabolism and feeding behavior.

<p>Two different experimental approaches were used to determine appetite response, relative meal volume and mean daily food intake. (A) Flies starved for 3 hours where transferred to synthetic food dyed with 0.01% Bromophenol blue. Three sets of (n = 100) flies were scored for blue positive abdomens over time. The starvation-induced appetite response was plotted as % of fly population that fed overtime. (B) Flies from A that scored positive at 2.5 hours were collected, homogenized and evaluated for Bromophenol blue content by measuring absorbance at 545 nm. Absorbance is proportional to relative meal volume and shown as the mean of three sets of (n = 25) flies. The capillary feeding (CAFE) assay was performed to determine (C) mean daily intake of liquid food media per day and (D) the number of meals consumed per hour, which were used to calculate (E) the average meal volume. Error bars are represented by the S.E.M. *p-values<0.05, **<0.01.</p

Caloric intake-independent obesity via regulation of Akhp cell viability and function.

<p>(A) Adipokinetic hormone-producing cells (Akhpc), (gfp) make up most of the corpus cardiac (cc) of the ring gland, located near the brain (dapi/blue = nuclear, nile red = counterstain). (B) <i>Akh</i> mRNA, which is only expressed in Akhp, is upregulated in <i>lace^k05305/2</i> and downregulated in <i>ifc⁴</i> mutants. (C) Fat mobilization in starved <i>ifc⁴</i> flies is incomplete before starvation-induced expiration, while no remaining TAG stores are detectable in <i>lace^k05305/2</i> flies. (D) Akhpc-specific RNAi mediated knockdown of <i>lace</i> and <i>ifc</i> phenocopy whole knockout <i>Akh</i> expression levels in 2 day old flies, with similar changes in (E) TAG level (control (AkhGal4/+;+) while Akhpc-specific overexpression of dIAP1 (inhibitor of apoptosis) mitigates the effects of <i>ifc</i> knockdown (Akh;UAS-<i>dIAP1</i>;UAS-<i>ifc</i>-rnai). These effects can be visualized in green (GFP) third instar larval Akhp cells, in Akhpc-specific (F) control (AkhGal4;UAS-GFP) (G) RNAi-mediated <i>lace</i> knockdowns (AkhGal4;UAS-<i>lace</i>-rnai), (H) RNAi-mediated <i>ifc</i> knockdowns (AkhGal4;UAS-<i>ifc</i>-rnai) (I) <i>dIAP1</i> overexpression (AkhGal4;UAS-<i>dIAP1</i>) and (J) dIAP rescue of ifc knockdowns (AkhGal4;UAS-<i>dIAP1</i>;UAS-<i>ifc</i>-rnai) Error bars are represented by the S.E.M.. *p-values<0.05, **<0.01.</p

Sphingolipid metabolism in flies and humans.

<p>(A) <i>De novo</i> sphingolipid metabolism begins with the condensation of serine with palmitoyl-CoA catalyzed by serine palmitoyl transferase, which is encoded by <i>lace</i> (1). The resultant ketone is rapidly reduced by the actions of 3-ketosphinganine reductase (2) into dihydrosphingosine (DHS). The addition of a second fatty acid chain is carried out by ceramide synthase, encoded by the gene <i>schlank</i> (3), to produce dihydroceramide (DHC). DHC is desaturated by sphingosine delta 4 desaturase, encoded by <i>ifc</i> (4), producing ceramide. Ceramide can then be degraded by the actions of ceramidase (5) to form sphingosine. Either sphingosine or DHS can be phosphorylated by Sphingosine kinase 1 or 2 (6). Once phosphorylated, either can be irreversibly degraded by the actions of S1P lyase, encoded by <i>Sply</i>. (B) Protein encoding genes of the SL pathway are highly conserved between flies and humans. Identity and E-value determined using pBLAST analysis of <i>Drosophila</i> proteins from Flybase against Human database. (NGI = No gene identified).</p