Hypoxia-inducible lipid droplet-associated interacts with DGAT1 and promotes lipid storage in hepatocytes
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Abstract
Lipid droplets (LD) are dynamic organelles that can expand and shrink, driven by fluctuations in the rate of triglyceride synthesis and degradation. Triglyceride synthesis, storage in LD, and degradation are governed by a complex set of LD-associated proteins. One of these LD-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair LD breakdown by inhibiting adipose triglyceride lipase. Here we characterized the physiological role and mechanism of action of HILPDA in hepatocytes. Expression of HILPDA was induced by fatty acids in several hepatoma cell lines. Hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglycerides in mice with non-alcoholic fatty liver disease. Similarly, deficiency of HILPDA in mouse precision-cut liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labelled fatty acids in LD, respectively, which was independent of adipose triglyceride lipase. Fluorescence microscopy showed that HILPDA partly colocalizes with LD and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging further revealed that HILPDA preferentially localizes to LD that are being remodelled. Mechanistically, HILPDA overexpression increased lipid storage in human hepatoma cells concomitant with an increase in DGAT activity and DGAT1 protein levels. Finally, confocal microscopy and Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA colocalizes and physically interacts with DGAT1. Overall, our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity. These findings suggest a novel mechanism in hepatocytes that links elevated fatty acid levels to stimulation of triglyceride synthesis and storage.
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###Reviewer #3:
Non-alcoholic fatty liver disease is a growing health issue worldwide. The pathogenesis and mechanism causing the disease are poorly understood. As the authors state correctly, unravelling mechanistic details of liver lipid metabolism is extremely important yet also technically very challenging. This report aimed at defining the role and mechanism of action of HILPDA in liver cells. The presented paper shows very interesting aspects on the role of HILPDA and brings novel concepts into the field and, as such, has extremely high potential. An overwhelming amount of data is shown that leads to development of the story. However, in the current form, the novel mechanism as outlined from the title has not been worked out with sufficient detail.
de la Rosa Rodriguez et al. claim that 'The increase of DGAT1 activity via HILPDA …
###Reviewer #3:
Non-alcoholic fatty liver disease is a growing health issue worldwide. The pathogenesis and mechanism causing the disease are poorly understood. As the authors state correctly, unravelling mechanistic details of liver lipid metabolism is extremely important yet also technically very challenging. This report aimed at defining the role and mechanism of action of HILPDA in liver cells. The presented paper shows very interesting aspects on the role of HILPDA and brings novel concepts into the field and, as such, has extremely high potential. An overwhelming amount of data is shown that leads to development of the story. However, in the current form, the novel mechanism as outlined from the title has not been worked out with sufficient detail.
de la Rosa Rodriguez et al. claim that 'The increase of DGAT1 activity via HILPDA is a novel mechanism that links elevated fatty acid levels to stimulation of triglyceride synthesis and storage in hepatocytes." Experiments correlate HILPDA with DGATs, e.g. upregulation of HILPDA in NASH, overexpression of HILPDA correlating with increase of DGAT1 levels, localization studies demonstrating colocalization of HILPDA with DGAT1 and DGAT2. As experienced in previous HILPDA studies, many effects are modest (e.g. decrease of TG in mice liver with NASH upon deletion of HILPDA, changes in plasma ALT levels).
As the authors correctly state in their results section, the presented data suggest that HILPDA promotes lipid storage at least partly via an ATGL-independent mechanism. Fig 3 also indicates different sized individual lipid droplets comparing Atglistatin treatment, even though the total LD area might differ significantly.
HILPDA is associated with increased DGAT activity, the suggested mechanism behind it (transcriptional activation?) is not described sufficiently. DGAT1 activity decreases FA-levels and as such would back in down-regulation HILPDA expression. To support the very interesting and very strong claim that DGAT1 is increased by direct interaction with HILPDA, this should be shown in vitro.
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###Reviewer #2:
This manuscript further characterizes the role of HILPDA/HIG2 in TAG/LD biology. The major finding is that HILPDA interacts with and promotes DGAT activity and TAG synthesis, which is novel given that HILPDA has largely been thought to regulate TAG turnover as a lipolytic inhibitor.
Characterization of the interaction between HILPDA and DGAT1 (and to a lesser extent DGAT2) is the major strength of this paper and an important advancement in the field. The early parts of the paper are not particularly novel (Fig. 1) or well-designed (Fig 2. - poor NAFLD/NASH model showing almost no effects) and the study is a bit on the thin side for data.
The data shown in Figure 1 is not particularly striking given that HILPDA is a known target gene of PPAR-alpha, which is activated by FAs. Showing that HILPDA expression tracks with …
###Reviewer #2:
This manuscript further characterizes the role of HILPDA/HIG2 in TAG/LD biology. The major finding is that HILPDA interacts with and promotes DGAT activity and TAG synthesis, which is novel given that HILPDA has largely been thought to regulate TAG turnover as a lipolytic inhibitor.
Characterization of the interaction between HILPDA and DGAT1 (and to a lesser extent DGAT2) is the major strength of this paper and an important advancement in the field. The early parts of the paper are not particularly novel (Fig. 1) or well-designed (Fig 2. - poor NAFLD/NASH model showing almost no effects) and the study is a bit on the thin side for data.
The data shown in Figure 1 is not particularly striking given that HILPDA is a known target gene of PPAR-alpha, which is activated by FAs. Showing that HILPDA expression tracks with PLIN2 is also pretty obvious as PLIN2 tracks with LD accumulation. I really don't see the need/relevance of this figure.
The MCD diet is widely regarded as a poor model for NAFLD/NASH since it doesn't replicate human NASH in so many regards. As a result, the use of this model makes these studies less relevant. Also, it is referenced that HILPDA was found to be up in a MCD study, but why not look at the plethora of human and mouse studies of NAFLD that have done RNAseq or arrays to provide a more physiological assessment of its expression in NAFLD/NASH?
The conclusion that effects are independent of ATGL are not overly convincing. Since ATGListatin is not specific for ATGL (Quiroga et al. 2018), a more thorough and quantitative analysis of TAG turnover with ATGL knockdown/out is warranted if these claims are to be made.
Since DGAT1 mRNA is unchanged but protein goes up, it would be assumed that HILPDA is affecting DGAT1 stability/turnover. This should be considered.
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###Reviewer #1:
This study dissects the role of LD associated protein HILPDA in triglyceride and LD homeostasis in hepatic tissue. Using a mouse tissue-specific HILPDA KO, live cell imaging, and lipid analysis, it proposes that HILPDA promotes TAG storage in LDs independently of ATGL regulation. Instead, HILPDA is proposed to interact with DGAT1 and promote TAG synthesis/storage.
This is an interesting and potentially exciting study that provides a new insight for HILPDA in liver fat storage. The proposed model differs from previous literature that proposes HILPDA regulates lipolysis via ATGL. Unfortunately, while the data presented support a potential role for HILPDA in DGAT regulation, a clear mechanism is not identified. The first half of the paper that phenotypes loss and over-expression of HILPDA is thorough and conclusive. The …
###Reviewer #1:
This study dissects the role of LD associated protein HILPDA in triglyceride and LD homeostasis in hepatic tissue. Using a mouse tissue-specific HILPDA KO, live cell imaging, and lipid analysis, it proposes that HILPDA promotes TAG storage in LDs independently of ATGL regulation. Instead, HILPDA is proposed to interact with DGAT1 and promote TAG synthesis/storage.
This is an interesting and potentially exciting study that provides a new insight for HILPDA in liver fat storage. The proposed model differs from previous literature that proposes HILPDA regulates lipolysis via ATGL. Unfortunately, while the data presented support a potential role for HILPDA in DGAT regulation, a clear mechanism is not identified. The first half of the paper that phenotypes loss and over-expression of HILPDA is thorough and conclusive. The latter half of the paper, investigating the interplay between HILPDA and DGAT1, appears more preliminary.
The critical issue in this study is that the nature of the HILPDA-DGAT1 interaction is not well defined. HILPDA over-expression is shown to increase DGAT1 protein levels, but the specific mechanism underlying this is not further dissected. Furthermore, it is still unclear whether this interaction is direct, or merely stochastic due to the fact that both DGAT1 and HILPDA reside on the same LDs in the experiments presented. More biochemical investigation as to whether these proteins physically interact in their native states, and if so whether that interaction affects DGAT1 enzymatic activity directly or allosterically, is required. Without this the study is mainly descriptive.
Major concerns:
Fig 4: overnight and acute fatty acid addition experiment: The authors propose that HILPDA enriches at sites where new fatty acids are being processed. Can you demonstrate that both these fluorescent FA species are even being incorporated into TAG during the time periods associated with the microscopy? An alternative explanation is simply that HILPDA localizes to regions of the cell where FA esterification or incorporation into other lipid species is occurring. TAG is potentially only one of many fates for these FAs. Can DGAT1/2 be colocalized with HILPDA in these experiments? Alternatively, what happens in these experiments if DGAT inhibitors are co-added with the FAs?
Fig 5H: The DGAT activity assays indicate that HILPDA over-expression increases the incorporation of fluorescent FA and DAG into TAG, but it is unclear as written whether these assays are normalizing for DGAT1 protein amount. Does HILPDA over-expression enhance DGAT enzymatic activity in this panel, or merely promote TAG synthesis here by the increased total DGAT protein level noted later in the study? This is a clear distinction in mechanism, and needs to be dissected further.
Fig 6/7: DGAT1-HILPDA interaction. The data presented in Fig 7 indicate that DGAT1 and HILPDA co-localize in cells and potentially are in very close proximity with one another. However, the data as presented are not enough to indicate whether these proteins directly interact. Do these proteins immunoprecipitate with one another? Some biochemical evidence for their interaction is necessary
Fig 7: relatedly, the mechanism by which DGAT1 is increased in protein level from HILPDA is also unclear. Is the protein more long-lived, or stabilized in the ER when HILPDA is over-expressed? Again, protein biochemical analysis would be helpful.
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##Preprint Review
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###Summary:
This study further characterizes the role of lipid droplet (LD) associated protein HILPDA in LD biology. The authors propose that HILPDA promotes triglyceride (TAG) storage in LDs by a mechanism independent of ATGL, through activation of DGAT. This is a potentially interesting finding, however, as detailed by the reviewers below, the data presented do not identify a mechanism for how HILPDA affects DGAT.
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