Discussion

Taken together total body fat storage is not changed in DmHsl¹ flies compared to control flies. A detailed analysis of different neutral lipid species by thin layer chromatography and lipidomics revealed no increase in total DAG species as observed in HSL^-/- mice (Haemmerle et al., 2002a). However, the substrate spectra of DmHsl and mouse HSL in in vitro assays are identical (personal communication from Dr. Christoph Heier and Dr. Robert Zimmermann [University Graz]) covering hydrolytic activities on: TAGs, DAGs (Fredrikson et al., 1986) (highest activity), MAGs (Fredrikson et al., 1981) and Cholesterol esters (Contreras et al., 1998). This would argue in favor of an evolutionary conserved function in DmHsl in flies. However, fly physiology differs from mammals as the main transport form of lipids in Drosophila are DAGs bound to lipoproteins (Palm et al., 2012). Therefore, it might be possible that there is a general redundancy of direct DAG lipolysis or a more flexible metabolism that allows a different processing of DAGs e.g conversion into a phospholipid and subsequent hydrolysis by specific phospholipases.

Shortly before I started my work on DmHsl the original DmHsl¹ flies (+ DmHsl^revertant and Act5c>GAL4 UAS-DmHsl) were sent (prepared by Iris Bickmeyer and Dr. Ronald Kühnlein) for a lipidomics analysis performed by the Lipidomics facility from Medical University Graz (used were 2x50 six-day-old flies, from seeding 150 embryos / midsize vial; from two independent density seedings). The analysis from the annotated lipid data revealed a significantly higher total TAG storage of control flies (DmHsl^revertant) compared to the DmHsl¹ and a ubiquitous (Act5c>GAL4) overexpression of Hsl (Figure

47). Total amounts of DAGs per fly were also highest in control flies and no significant differences could be observed between Hsl overexpressing of deficient flies (Figure 47). Nevertheless, the % of DAGs from total TAGs indicated a significant increase in

% of DAGs of total lipids in DmHsl¹ and gof-DmHsl flies (Figure 47). No differences could be detected between the mutant and overexpression flies indicating that there is no significant increase DAG species in DmHsl¹ flies. The observed differences are rather a results of a sub-optimal matched control for the DmHsl¹ stock as TAG amounts in backcrossed flies did not differ between DmHsl¹ and controls (Figure 47).

A TLC analysis (performed by Iris Bickmeyer) of flies used for the lipidomics analysis revealed differences in TAG storage but not in DAGs (data not shown).

Figure 47 Preliminary results indicate that DmHsl¹ flies do not accumulate diacylglycerol. A lipidomic analysis performed by the Lipidomics facility of the Medical University Graz using non-backcrossed DmHsl¹ flies (6d males, prepared by Iris Bickmeyer and Dr. Ronald Kühnlein) showed a small but significant increase in %DAGs from total detected TAGs (C). Absolute amounts of TAGs and DAGs were significantly higher in control flies compared to DmHsl¹ but flies ubiquitously over expressing DmHsl (Act5c>GAL4) showed comparable total amounts. A strong accumulation of DAGs cannot be seen (consistent with TLC data). Plotted are the means of average lipid amounts of all detected TAG and DAG species detected per male flies (A,B) and calculated % of DAGs (C) of total TAGs (±SEM; Student’s t-test, *=P<0.05).

As fed flies were used for the TLC experiment as well as for the Lipidomics analysis (non-backcrossed DmHsl¹ stock was analyzed) a possible effect of DAG accumulations might be too mild in order to be detected. On the other hand, lipid mobilization in DmHsl¹ flies was indifferent from control flies. The TLC analysis from abdomen and thorax samples exhibited a different lipid species profile with a strong enrichment in a lipid with a comparable running behavior as DAGs on the TLC plate. Though a difference between DmHsl¹ and control flies could not be detected. Conversely, an

overexpression of DmHsl::egfp exhibits a trend towards decreased body fat indicating a involvement in lipolysis of DmHsl.

Comparable to studies in larvae (Bi et al., 2012) overexpressed DmHsl::egfp also showed in adult fat body tissue increased abundance on LDs under starvation. A general absence of overexpressed DmHsl::GFP on LDs in starved plin1¹ flies could not be seen but large lipid droplets were omitted. Whether this is caused by the absence of Plin1 directly, due to a missing interaction partner for DmHsl or an indirect effect caused by changed physicochemical properties of large LDs remains to be answered.

Though overexpressed DmHsl::egfp was used for in vivo localization studies in larvae (Bi et al., 2012) and adults, DmHsl could also be found on embryonic LDs (Cermelli et al., 2006) and on induced LDs in S2 cells (Krahmer et al., 2013). Apart from that DmHsl was not found on LDs in fed larvae (Beller et al., 2006, Sahu-Osen, 2015). As embryogenesis represents a starvation state and the larval stage a feeding state a conserved mechanism of DmHsl localizing to lipid droplets under catabolic conditions might explain the finding and absence in the different lipid droplet proteomic studies.

The high maternal mRNA contribution of DmHsl as well as the detection of DmHsl on embryonic LDs implied a possible impairment in fertility of DmHsl¹ flies. DmHsl¹ flies showed no noticeable difference during general stock keeping compared to control flies. Consistently, a fecundity assay revealed no changes between control and DmHsl¹ flies. Although male HSL^-/- mice were sterile due to gonadal hypotrophy and oligospermia (Osuga et al., 2000), DmHsl¹ male flies generated similar numbers of offspring with DmHsl¹ and control females. However, fecundity was analyzed under laboratory terms providing ideal conditions for propagation. Therefore, a DmHsl deficiency might only be detrimental under wildtype living conditions. Also, the data indicates again a possible redundancy of DmHsl function as, despite the maternal DmHsl mRNA contribution, a DmHsl deficiency does not affect survival rate of embryos significantly.

When comparing the two available DmHsldeficient fly stocks (DmHsl¹ and DmHsl^b24) possible differences should be addressed. Of course studies on DmHsl^b24 mutants were restricted to larvae but these mutants showed TAG mobilization defects as well

as increased body fat storage in L3 larvae (Bi et al., 2012). Both effects could not be detected in DmHsl¹ adults. Therefore, the DmHsl¹ data should be verified with DmHsl^b24 flies and a proper genetically matched control for this strain.

In summary, DmHsl¹ flies are homozygous viable and show no obvious alterations in lipid storage and mobilization which might be compensated by so far undetected proteins. Interestingly, no homolog for hormone-sensitive lipase homolog can be found in birds suggesting also an alternative way to mobilize DAGs.

6 Supplement 2