6. Discussion
6.1. DGAT1 primarily facilitates murine hepatic MCFA incorporation into TAG
Although DGAT1 and DGAT2 seem to have different physiological functions, which is reflected by the different phenotypes of their knockout mouse models, the ability of hepatic DGAT1 and DGAT2 to partially compensate for each other in TAG synthesis, has been shown in previous studies (Smith et al., 2000; Stone et al., 2004; Li et al., 2015). In this work, the compensating ability of DGAT enzymes was analyzed in more depth, focusing on the analysis of chain length and saturation preferences of each DGAT enzyme, using different DGAT inhibitors and the DGAT1-/- mouse model combined with MS-based alkyne lipid tracing with species resolution.
As already mentioned, there are various studies, dealing with acyl-chain length and saturation preferences from each DGAT enzyme. Studies working with mammalian and fungal DGAT enzymes proclaim a preference of DGAT2 for medium-chain CoAs, whereas in many plant species DGAT1 isoforms are associated with preference for medium-chain CoAs in seed oil production (Lardizabal et al., 2001; Aymé et al., 2015; Iskandarov et al., 2017;
Klizaite, 2017). In the experiments presented in chapter 5.1 of this study using DGAT1 and DGAT2 inhibitors during an C19/FA co-incubation, showed a drastic reduction of C11-FA incorporation into TAG upon DGAT1 inhibition, whereas C19-C11-FA incorporation was slightly elevated (Figure 11). Upon DGAT2 inhibition, the vice versa effect could be observed, whereas a combination of both inhibitors nearly abolished MCT production with a corresponding accumulation of DAGs (Figure 11). These observations from the TLC experiments already suggested a DGAT1 preference for MCFA in hepatocytes.
Using the newly developed MS-based detection system for alkyne lipids, similar experiments were conducted, utilizing only C17:2 for tracing aTAG levels upon DGAT inhibitory treatment.
For overall aTAG levels, the MS experiments did show comparable results to these obtained from TLC analysis (Figure 22 A, B). The detailed NFC array analysis, however, revealed a complex pattern of up- and downregulated aTAG species upon inhibitory treatment. The proposed acyl-chain length preference for both DGATs was confirmed, as all shorter chain aTAG species showed a strong downregulation upon DGAT1 inhibition and a strong upregulation upon DGAT2 inhibition (Figure 23). Additionally, this dataset shows a tendency towards saturation preferences of each DGAT enzyme. DGAT1 seems to prefer more saturated species in the LCFA-aTAGs, as upon inhibition, the most saturated members in this group were downregulated. A combination of both inhibitors led to an even further
downregulation of aMCTs with a few strongly upregulated aLCT species. The latter correspond to TAGs synthetized from the most abundant FA species in murine hepatocytes, namely palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0) or oleic acid (C18:1) (Kindt et al., 2018). This may reflect a compensating effect of the remaining DGAT activity, maintaining FFA homeostasis by metabolizing DAGs which originate from remodeling of phospholipids, which contain mainly the FAs mentioned above.
In DGAT1-/- hepatocytes, slightly reduced aMCTs and increased aLCTs were observed while overall aTAG levels were similar (Figure 20 A, Figure 21 A, Supplementary figure 10, lanes labeled neg). As both cell types were fed with C17:2-FA and C10-FA, differences due to a lack of C10-CoA can be ruled out. The analogous TLC-experiments also showed slightly reduced C11-TAG levels in comparison to C19-TAG levels (Figure 26 B, data not quantified).
Upon DGAT2 inhibition, a TAG phenotype like the one in WT hepatocytes where both DGATs were inhibited could be reproduced. aMCT levels were further reduced as well (Figure 24 B, 25 B, 26 B/D). As no DGAT1 was present, this suggests, that the compensating ability of DGAT2 for DGAT1 includes a certain flexibility in DGAT2’s preference for acyl-CoAs, including those predominantly preferred by DGAT1. It is known, that DGAT2 locates both to the ER and LDs, whereas DGAT1 only locates to the ER (Kuerschner et al., 2008; Stone et al., 2009; Wurie et al., 2011). DGAT2 is also described to have a broader acyl-CoA specificity (Yen et al., 2008). The study of Li and colleagues suggests that the function of murine hepatic DGAT1 is mainly to channel FAs into TAG, which are then prone to be β-oxidized (Li et al., 2015). Altogether this may suggest, that hepatic DGAT1 acts mostly on MCFAs and that the DGAT2 population located at the ER is solely responsible for compensating the missing DGAT1 activity in DGAT1-/- hepatocytes.
Thus, the pressure on the system created through additional DGAT2 inhibition may be reduced by direct oxidation of the acyl-CoAs without channeling them through a TAG pool first, resulting in further decrease of MCT levels. As hepatic β-oxidation of MCFAs was shown to be very abundant, the mechanisms mentioned above provide a possible explanation (Pégorier et al., 1988; Metges and Wolfram, 1991). Similar effects in DGAT1 -/-hepatocytes were observed upon Etomoxir treatment, which are discussed in chapter 6.2.
Also, the pulse-chase experiments presented in chapter 5.3.1. showed an DGAT1- dependent incorporation of MCFAs into TAG. As these experiments were also including a comparison to Etomoxir-treatment, they are discussed in chapter 6.2.
In all experiments conducted with the DGAT inhibitors in WT hepatocytes, either within the TLC-based or the MS-based experiments, overall effects on aTAG levels were always more prominent upon DGAT1- rather than DGAT2-inhibition. A study in HepG2 cells, showed that DGAT1 is responsible for about 80% of overall TAG synthesis (Qi et al., 2012). This might serve as a possible explanation for the stronger overall effects on TAG levels upon DGAT1
Discussion
inhibition, presented in this thesis. However, as the metabolic properties of HepG2 cells are not directly comparable to those of primary hepatocytes this interpretation should be considered with caution. It also has been proposed, that DGAT1 predominantly acts on FA from exogenous sources, whereas DGAT2 mainly utilizes FA from de novo synthesis (Villanueva et al., 2009; Qi et al., 2012). As the hepatocytes were always exogenously fed with fatty acids, one would expect a more drastic effect upon DGAT1 inhibition. However, as already mentioned, the experiments in DGAT1-/- hepatocytes, showed similar overall concentrations of aTAG. Also considering the fact that in DGAT1-/- hepatocytes, the overall labeled lipid concentrations were similar to those in WT controls (Supplementary figure 8), it may be postulated that DGAT2 can, at least in murine hepatocytes, fully compensate for the loss of DGAT1, to maintain the overall TAG levels.
Changes in levels of aPC and aPA upon inhibitor treatment were visible as well (Supplementary Figure 9). aPA levels were significantly reduced upon either inhibitor treatment, both in WT and DGAT1-/- hepatocytes. This may be due to a positive feedback loop from accumulating DAG, which triggers a faster channeling of PA to form PI and PG. As the reduced aPA was also visible upon DGAT1 inhibitory treatment in DGAT1-/- hepatocytes it may represent an off-target effect form the DGAT1 inhibitor affecting other enzymes upstream of PA. However, as aPC and aDAG levels were not altered significantly in DGAT1-/- hepatocytes upon DGAT1 inhibition (Figure 24 B, Supplementary Figure 9), it is not possible to proof this hypothesis. aPC levels were significantly increased in WT cells upon DGAT1 inhibition, but in DGAT1-/- hepatocytes only upon DGAT2 inhibition. This is most likely due to the highest increase of DAG levels upon the strongest DGAT inhibition in each condition, and therefore excess DAG is as well channeled into PC synthesis.