Lipid mobilization in Drosophila melanogaster

In order to mobilize lipids Drosophila has a system that works similar to β-adrenergic signaling in mammals, the Adipokinetic hormone (Akh)-signaling pathway (Patel et al., 2005, Grönke et al., 2007, Galikova et al., 2015). Akh is a short neuropeptide of eight amino acids that interacts specifically with the Akh receptor (AkhR) leading to lipid mobilization (Lee and Park, 2004, Galikova et al., 2015). Akh is exclusively expressed in the corpora cardiaca (a portion of the ring gland) the major neuroendocrine organ in insects (Stone et al., 1976, Noyes et al., 1995) and Akh secretion is controlled by the extracellular trehalose concentration (Rulifson et al., 2002). Interestingly, whereas Akh deficiency had no effect on ontogenesis, locomotion, oogenesis, lipid- and carbohydrate storage until the end of metamorphosis, in adults Akh regulates body fat as well as hemolymph sugar levels (Galikova et al., 2015). Comparable to a AkhR¹ mutant flies, also Akh^A mutants have increased lipid storage but glycogen stores were normal. Consistently, both mutants have a higher starvation resistance. However, lipids were still mobilized under starvation in both mutants, indicating a second system that stimulates lipid mobilization (Grönke et al., 2007, Galikova et al., 2015).

With the binding of Akh to the AkhR in a target tissue like the Drosophila fat body it activates this GPCR leading to the activation of the G protein α q subunit (Gαq), G protein γ1 (Gγ1) and Phospholipase C at 21C (Plc21C) and subsequently increasing the intracellular Ca²⁺ (iCa²⁺) and cAMP concentrations (Figure 9). An RNAi mediated knock down of AkhR, Gαq, Gγ1 and Plc21C leads to decreased iCa²⁺ levels (Baumbach et al., 2014b) and finally resulted in increased lipid storage in the fat body. The same effect could also be observed by a knockdown of the stromal interaction molecule (Stim) or Inositol-1,4,5,-tris-phosphate (IP3) receptor (Itpr83A/DmITPR) (Baumbach et al., 2014a).

Downstream of Akh-signalling we can find a similar pattern to mammals for the lipid mobilization (Figure 9). The activated PKA leads to the phosphorylation of DmPlin1 (Patel et al., 2005) which leads to elevated lipolytic activity (Arrese and Wells, 1994).

Plin1 can be found primarily on larger “mature” lipid droplets (Beller et al., 2010) and expression as well as translation is tightly correlated with the cumulative LD surface

area and thereby adjusts to the total fat storage in the fly (Beller et al., 2010). plin1¹ mutant flies have increased fat storage and a giant lipid droplet phenotype (LD diameter >30μm). However, plin1¹ mutant flies still can mobilize storage lipids and have a higher starvation resistance than control flies. This indicates that plin1 is not crucial for lipid mobilization but an important member of the AkhR-dependent lipolysis pathway (Beller et al., 2010).

In Drosophila also a homolog of mammalian ATGL can be found namely Brummer (encoded by brummer or bmm). bmm knock out mutant (bmm¹) flies are obese whereas an overexpression leads to decreased body fat storage in adult flies (Grönke et al., 2005). Like its mammalian relative that acts as the main TAG lipase in adipose tissue (Smirnova et al., 2006) also Bmm catalyses the hydrolysis of TAGs to DAGs in flies but does not processes DAGs or MAGs (Grönke et al., 2005). bmm expression correlates with the feeding state of the fly and is upregulated under nutrient deprivation (Grönke et al., 2005). Like AkhR¹ mutants also bmm¹ mutant flies are still capable to mobilize lipids during starvation. On the other hand, AkhR¹ bmm¹ double mutant as well as bmm¹ plin1¹ double mutant flies cannot mobilize lipids anymore and exhibit severe obesity (Grönke et al., 2007).

Figure 9 Schematic overview of storage lipid mobilization in Drosophila melanogaster for ATP synthesis. Adipokinetic hormone (Akh) binding to the Akh-Receptor (AkhR) induces a signal cascade that activates PKA and increases intracellular Ca²⁺ levels and subsequently activating cAMP responsive element binding protein (CREBP). The main triglyceride lipase Brummer lipase performs TAG hydrolysis. Phosphorylated Plin1 sequesters Hsl to lipid droplets to further catalyse storage lipid hydrolysis. The released fatty acids (NEFAs) are directed towards β-oxidation in peroxisomes (especially very-long and long chain fatty acids (VLCFAs and LCFAs) and mitochondria (here imported via carnitine-shuttle with the rate limiting step catalysed by Carnitine palmitoyltransferase I (CPT1)).

Acetyl-CoA enters TCA and finally ATP is generated by oxidative phosphorylation (OXPHOS). Hepatic nuclear factor 4 (HNF4) can be activated by fatty acids and finally and improves cellular substrate oxidation and respiration.

However, it was not known if we can find also proteins with HSL and ABHD5 function and/or sequence similarity in the fly to see if the whole lipid mobilization pathway is evolutionary conserved. Indeed, unique sequence related proteins for HSL (Grönke, 2005) and ABHD5 (Takacs, 2007) could be found also in the fly namely Hormone-sensitive lipase ortholog (encoded by Hsl, CG11055) and Pummelig (encoded by puml, CG1882). Moreover, mutants had been generated for both genes in previous works (Grönke, 2005, Takacs, 2007).

DmHsl is expressed during all developmental stages with a strong enrichment in early embryogenesis indicating a strong maternal contribution (Grönke, 2005, Bi et al., 2012). Hsl¹ mutant flies have normal body fat (Grönke, 2005), as so have HSL

-/-knockout mice (Haemmerle et al., 2002a). At the same time, starvation resistance of DmHsl¹ flies was not significantly different to control flies (Grönke, 2005).

Recently after I started with the further characterization of the DmHsl¹ mutants a different group published data on DmHsl (Bi et al., 2012).

In larvae, DmHsl (as well as bmm) expression is upregulated during starvation (Bi et al., 2012). Bi and colleagues also generated an independent knockout mutant called DmHsl^b24 that was used in their experiments. The DmHsl^b24mutant larvae exhibited increased glyceride storage (+30%) and Bi et al. claimed that the mutant larvae had lipid mobilization defects. At least the larval lipid over-storage phenotype was incoherent with normal fat storage of DmHsl¹ mutant flies. Hence, it might be possible that the over-storage phenotype is restricted to the larval stage.

A fat body specific overexpression of DmHsl::eGFP in larvae showed the same characteristics as mammalian HSL. While being localized in the cytoplasm under basal conditions, abundance of DmHsl::eGFP on lipid droplets was increased during starvation (Bi et al., 2012). Additionally, fat body overexpressed DmHsl::eGFP was unable to localize on Lipid droplets in plin1¹ larvae, leading to the assumption that, like in mammals, Plin1 sequesters DmHsl::eGFP onto the LD surface (Bi et al., 2012).

However, this studies focused mainly on larvae and indicated an evolutionary conserved function of DmHsl to mammalian HSL (Grönke, 2005, Bi et al., 2012).

Therefore, DmHsl was analysed further to characterize its function in adult flies and identify a possible biological phenotype in DmHsl¹ flies (see Supplement).

1.3.2 Pummelig the single sequence related protein to mammalian α/β-hydrolase