InteractiveFly: GeneBrief

Phosphoethanolamine cytidylyltransferase: Biological Overview | References

Diet-induced obesity leads to dysfunctional feeding behavior. However, the precise molecular nodes underlying diet-induced feeding motivation dysregulation are poorly understood. Using a longitudinal high-sugar regime in Drosophila, this study sought to address how diet-induced changes in adipocyte lipid composition regulate feeding behavior. It was observed that subjecting adult Drosophila to a prolonged high-sugar diet degrades the hunger-driven feeding response. Lipidomics analysis reveals that longitudinal exposure to high-sugar diets significantly alters whole-body phospholipid profiles. By performing a systematic genetic screen for phospholipid enzymes in adult fly adipocytes, Phosphoethanolamine cytidylyltransferase (Pect) was identified as a critical regulator of hunger-driven feeding. Pect is a rate-limiting enzyme in the phosphatidylethanolamine (PE) biosynthesis pathway and the fly ortholog of human PCYT2. Disrupting Pect activity only in the Drosophila fat cells causes insulin resistance, dysregulated lipoprotein delivery to the brain, and a loss of hunger-driven feeding. Previously human studies have noted a correlation between PCYT2/Pect levels and clinical obesity. Now, these unbiased studies in Drosophila provide causative evidence for adipocyte Pect function in metabolic homeostasis. Altogether, this study has uncovered that PE phospholipid homeostasis regulates hunger response (Kelly, 2022).

Improper hunger-sensing underlies a multitude of eating disorders, including obesity. Yet, the cellular and molecular mechanisms governing the breakdown of the hunger-sensing system are poorly understood. In addition to lipid storage, adipocytes play a crucial endocrine role in maintaining energy homeostasis. Factors secreted by adipocytes impinge on several organs, including the brain, to regulate systemic metabolism and feeding behavior. Since lipids play a key role in signaling, adipocyte lipid composition is likely to regulate hunger perception and feeding behavior. Linking specific changes in adipocyte lipid composition to hunger perception and feeding behavior remains challenging (Kelly, 2022).

While the effects of neutral fat reserves such as triglycerides on feeding behavior have been extensively studied, less is known about the effects of phospholipids. Phospholipids comprise the lipid bilayer of the plasma membrane and anchor integral membrane proteins, including ion channels and receptors. They are essential components of cellular organelles, lipoproteins, and secretory vesicles. Changes to phospholipid composition can alter the permeability of cell membranes and disrupt intra- and intercellular signaling. Numerous clinical studies suggest an association between phospholipid composition and obesity. For example, insulin resistance, a hallmark of obesity-induced type 2 diabetes, is strongly associated with alterations in phospholipid composition. Additionally, key phospholipid biosynthesis enzymes are correlated with obesity in human genome-wide association studies. Despite these intriguing possibilities, a causative link between altered phospholipid composition and metabolic dysfunction is yet to be established. Furthermore, whether altered adipocyte phospholipid composition specifically leads to dysfunctional hunger-sensing is unknown (Kelly, 2022).

Phosphatidylethanolamine (PE) is the second most abundant phospholipid and is essential in membrane fission/fusion events. PE is synthesized through two main pathways in the endoplasmic reticulum (ER) and the mitochondria. Phosphatidylethanolamine cytidylyltransferase (Pcyt/Pect) is the rate-limiting enzyme of the ER-mediated PE biosynthesis pathway (Dobrosotskaya, 2002). Global dysregulation in Pcyt/Pect activity has been shown to cause metabolic dysfunction in animal models and humans (Lim, 2011; Tsai, 2019). For example, Pyct/Pect deficiency in mice causes a reduction in PE levels, leading to obesity and insulin resistance (Fullerton, 2009). Similarly, human studies have found that obese individuals with insulin resistance have decreased Pcyt/Pect expression levels. Chronic exposure to a high-fat diet causes upregulation of Pcyt/Pect, associated with increased weight gain and insulin resistance (de Wit, 2008). These findings suggest that disruptions in Pcyt/Pect activity, and consequently PE homeostasis, are a common underlying feature of obesity and metabolic disorders. What remains largely unknown is whether Pcyt/Pect activity in the adipose tissue directly regulates insulin sensitivity and feeding behavior (Kelly, 2022).

Like humans, chronic overconsumption of a high-sugar diet (HSD) results in insulin resistance, diet-induced obesity (DIO), and metabolic imbalance in flies. There is deep evolutionary conservation of feeding neural circuits regulating feeding behavior between flies and mammals, and multiple studies on feeding behavior in Drosophila have identified key neurons and receptors involved. Furthermore, like humans, Drosophila display altered feeding behavior in response to highly palatable foods. Additionally, given flies' short lifespan, feeding behavior in response to an obesogenic diet can be monitored throughout the adult fly's lifespan, providing temporal resolution of behavioral changes under DIO. Thus, using a chronic HSD feeding regime in adult flies allows for discovering specific mechanisms relevant to human biology (Kelly, 2022).

This study assesses the effects of chronic HSD consumption on flies' hunger-driven feeding (HDF) behavior across a 28-day time window. It is noted that while HSD-fed flies maintain their ability to mobilize fat stores on starvation, they lose their HDF response after 2 weeks of HSD treatment, suggesting an uncoupling of nutrient sensing and feeding behavior. This study revealed that changes in phospholipid concentrations in HSD-fed flies occur during HDF loss. It was further shown that genetic disruption of the key PE biosynthesis enzyme Pect in the fat body, the fly's adipose tissue, results in the loss of HDF even under normal food (NF) conditions. Significantly, Pect overexpression in the fat body is sufficient to protect flies from HSD-induced loss in HDF. These data suggest that adipocyte PE-phospholipid homeostasis is critical to maintaining insulin sensitivity and regulating hunger response (Kelly, 2022).

Several studies have shown a link between chronic sugar consumption and altered hunger perception. Although the neuronal circuits governing hunger and HDF behavior have been well studied, less is known about the impact of adipose tissue dysfunction on feeding behavior. Using a Drosophila DIO model, this study showed that phospholipids, specifically PE, play a crucial role in maintaining HDF behavior (Kelly, 2022).

The Drosophila model organism is a relevant model for human DIO and insulin resistance. Previous studies have performed measurements on taste preference, feeding behavior/intake, survival, etc., using an HSD-induced obesity model, and have found much in common with their mammalian counterparts. However, the longest measurement of adult feeding behavior has been capped at 7 days. A recent study by analyzed the fly lipidome on 3-week and 5-week HSD in a tissue-specific manner and identified changes in neutral fat stores in the cardiac tissue (Kelly, 2022).

This study defined that a 14-day exposure of adult Drosophila to an HSD regime disrupts hunger response. On evaluating HSD regime-induced lipid composition changes at this critical 14-day point, a critical requirement was uncovered for adipocyte PE homeostasis and a fat-specific role for the PE enzyme Pect in controlling HDF. Pect function in the adult fly adipocytes is critical for appropriate fat-to-brain lipoprotein delivery and the maintenance of systemic insulin sensitivity. In sum, this study identified that adipocyte-specific loss of Pect phenocopies the metabolic dysfunctions observed in a chronic HSD regime in adult flies. Therefore, it is proposed that PE homeostasis, specifically Pect activity in fat tissue, regulates HDF response (Kelly, 2022).

Changes in feeding behavior in both vertebrates and invertebrates occur via communication between peripheral organs responsible for digestion/energy storage and the brain. This communication is facilitated by factors that provide information on nutritional state. One example of such a factor is leptin, released from the adipose tissue and acts on neuronal circuits in the brain to promote satiety. While leptin has long been studied as a satiety hormone, recent work in mice and flies suggests that a key function of leptin and its fly homolog upd2 regulates starvation response. Indeed, previous work has shown that exposing flies to HSD alters synaptic contacts between Leptin/Upd2 sensing neurons and Insulin neurons. However, it resets within 5 days, suggesting that yet-to-be-defined mechanisms maintain homeostasis on surplus HSDs beyond 5 days (Kelly, 2022).

Feeding behavior was analyzed over time to delineate how HSD alters the starvation response.under normal diet conditions flies display a clear response to starvation in the form of elevated feeding that is termed 'hunger-driven feeding (HDF),' which was independent of age. In contrast, chronic exposure to HSD led to a progressive loss of HDF that began on day 14. It could be argued that loss of HDF is simply due to an elevation of TAG storage in HSD-fed flies, thus losing the need to feed on starvation. However, several pieces of evidence support the idea that HSD affects feeding behavior independently of nutrient sensing. Under the current experimental conditions, this study found basal feeding to be statistically similar between NF-fed and HSD-fed conditions at all timepoints with the exception of day 10. Note that it has been reported that on a 20% sucrose liquid diet for 7 days elevated food interactions. However, those studies are not comparable with the current study due to the large differences in experimental protocol. The previous study evaluated taste preference changes and feeding interactions on 5-30% sucrose liquid diet in 24-hr window over a period of 7 days. This study assessed food interaction in a 3-hr window, after providing a complex lab standard diet, to monitor HDF. Future studies would be needed to assess the effect of 14-day HSD on taste perception using the experimental design in this study. The HDF response of HSD-fed flies is significantly lower than that of NF-fed flies, but they sense energy deficit and mobilize fat stores accordingly. Hence, HSD-fed flies can calibrate their HDF to compensate only for the amount of fat lost in starvation. Nonetheless, this capacity of flies to couple energy sensing and feeding motivation is lost beyond day 14, as evidenced by the loss of HDF and continuous TAG breakdown. Strikingly, subjecting 14-day HSD-fed flies to prolonged starvation (up to 32 hr) was insufficient to induce increased HDF. While there was an uptick in feeding behavior at 20 hr of starvation, this hunger response was not sustained at 24 and 32 hr, even though flies continued to mobilize TAG reserves at 24 and 32 hr. Thus, prolonged exposure to HSD leads to uncoupling nutrient sensing and feeding behavior (Kelly, 2022).

Notably, fly and mammalian DIO models have striking differences and similarities. Mice show linear weight gain on obesogenic diets, but flies' rigid exoskeleton limits their capacity to store TAG beyond a certain point. However, similar to mammals, prolonged exposure to HSD, strongly associated with phospholipid dysregulation, leads to reduced insulin sensitivity. This study shows that the levels of Dilp5, the fly's insulin ortholog, are reduced in the IPCs of HSD-fed flies. However, no decrease in Dilp5 or Dilp2 mRNA levels was observed; this is suggestive of increased insulin secretion on HSD, similar to previously reported. Consistent with the idea that 14-day HSD triggers insulin resistance, elevated FOXO nuclear localization was observed in the fat bodies of the HSD-fed flies, despite a likely increase in Dilp5 secretion on HSD. Again, these findings align with mammalian studies showing that dysregulated FOXO signaling is implicated in insulin resistance, type 2 diabetes, and obesity (Kelly, 2022).

Changes in the lipidome are strongly correlated with insulin resistance and obesity . However, less is known about how the lipidome affects feeding behavior. To this end, the lipid profiles of NF and HSD-fed flies were examined over time. As expected, exposure to HSD increased the overall content of neutral lipids compared to the NF flies, with TAGs and DAGs increasing the most, which is consistent with other DIO models. Surprisingly, it was noted that 14 days of HSD treatment caused a decrease in FFAs and a rise in TAGs and DAGs. It is speculated that this reduction in FFA may be due to their involvement in TAG biogenesis. It was of interest to see whether the decrease in FFA correlated to a particular lipid species as PE and PC are made from DAGs with specific fatty acid chains. However, further analysis of FFAs at the species level did not reveal any distinct patterns. Most FFA chains decreased in HSD, including 12.0, 16.0, 16.1, 18.0, 18.1, and 18.2. This data was more suggestive of a global decrease in FFA, likely converted to TAG and DAG rather than depleting a specific fatty acid chain (Kelly, 2022).

On day 14 of HSD treatment, when HDF response begins to degrade, PE and PC levels rise dramatically, whereas LPE significantly decreases. Interestingly, similar patterns of phospholipid changes have been associated with diabetes, obesity, and insulin resistance in clinical studie, yet no causative relationship has been established. Intriguingly, this study found that PC balance appears dispensable for maintaining HDF-response. But both the mitochondrial and cytosolic PE pathways seem critical for HDF response. Multiple pathways synthesize PE. Studies have shown that in addition to the mitochondrial PISD and cytosolic CDP-ethanolamine Kennedy pathway, PE can be synthesized from LPE. This pathway is named the exogenous lysolipid metabolism (ELM) pathway. ELM can substitute for the loss of the PISD pathway in yeast and requires the activity of the enzyme lyso-PE acyltransferase (LPEAT) that converts LPE to PE. In this study, it is noted PE levels were upregulated on HSD while LPE levels were downregulated (Kelly, 2022).

In contrast, fat-specific Pect-KD caused PE levels to trend downward, whereas LPE was upregulated. Though the level changes for PE and LPE are contrasting between 14-day HSD lipidome and Pect-KD, under both states, there is an imbalance of phospholipids classes PE and LPE. Hence, it is propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to HDF and insulin sensitivity (Kelly, 2022).

The role of the minor phospholipid class LPE remains obscure. This study observes that the LPE imbalance occurs during prolonged HSD exposure and when fat body Pect activity is disrupted. This suggests that LPE balance likely plays a role in insulin sensitivity and the regulation of feeding behavior. It is anticipated that this observation will stimulate interest in studying this poorly understood minor phospholipid class. In future work, it would be interesting to test how the genetic interactions between the enzyme that converts LPE to PE, called LPEAT, and Pect manifest in HDF. Specifically, it will be interesting to ask whether reducing or increasing LPEAT will restore PE-LPE balance to improve the HDF response in HSD-fed flies and Pect-KD. Future studies should explore how LPE-PE balance can be manipulated to affect feeding behaviors (Kelly, 2022).

In addition to changes in phospholipid classes, this study found that HSD caused an increase in the concentration of PE and PC species with double bonds. Double bonds create kinks in the lipid bilayer, leading to increased lipid membrane fluidity, impacting vesicle budding, endocytosis, and molecular transport. Hence, a possible mechanism by which HSD induces changes to signaling by altering the membrane biophysical properties, such as by increased fluidity; this would impact various cellular processes, including synaptic firing and inter-organization vesicle transport. Consistent with this idea, a significant reduction was observed in the trafficking of ApoII-positive lipophorin particles from adipose tissue to the brain. Targeted experiments are required to understand how lipid membrane fluidity alters hunger response fully (Kelly, 2022).

To explore the idea that fat-brain communication may be perturbed under HSD and Pect knockdown, a fat-specific signal known to travel to the brain was examined. ApoLpp chaperones PE-rich vehicles called lipophorins traffic lipids from fat to all peripheral tissues, including the brain. ApoII, the Apolpp fragment harboring the lipid-binding domain, has been shown to regulate systemic insulin signaling by acting on a subset of neurons in the brain. This study found that both HSD treatment and Pect knockdown reduced ApoII levels in the brain. Given that ApoII acts as a ligand for lipophorin receptors in the brain, ApoII may be a direct regulator of feeding. Alternatively, it could ferry signaling molecules and PE/PC lipids. In the future, it would be important to explore whether lipoprotein trafficking from fat-to-brain directly impacts the hunger response (Kelly, 2022).

This study has uncovered a role for the phospholipid enzyme Pect as an important component in maintaining HDF. Future work should explore the precise mechanism of how Pect and the associated disruption in phospholipid homeostasis can impact adipose tissue signaling. In sum, this study lays the groundwork for further investigation into Pyct2/Pect as a potential therapeutic target for obesity and its associated comorbidities (Kelly, 2022).

PE homeostasis rebalanced through mitochondria-ER lipid exchange prevents retinal degeneration in Drosophila

The major glycerophospholipid phosphatidylethanolamine (PE) in the nervous system is essential for neural development and function. There are two major PE synthesis pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum (ER) and the phosphatidylserine decarboxylase (PSD) pathway in mitochondria. However, the role played by mitochondrial PE synthesis in maintaining cellular PE homeostasis is unknown. This study shows that Drosophila pect (phosphoethanolamine cytidylyltransferase) mutants lacking the CDP-ethanolamine pathway, exhibited alterations in phospholipid composition, defective phototransduction, and retinal degeneration. Induction of the PSD pathway fully restored levels and composition of cellular PE, thus rescued the retinal degeneration and defective visual responses in pect mutants. Disrupting lipid exchange between mitochondria and ER blocked the ability of PSD to rescue pect mutant phenotypes. These findings provide direct evidence that the synthesis of PE in mitochondria contributes to cellular PE homeostasis, and suggest the induction of mitochondrial PE synthesis as a promising therapeutic approach for disorders associated with PE deficiency (Zhou, 2020).

In a forward genetic screen to identify genes necessary for photoreceptor cell survival, this study isolated mutations in the gene pect, which encodes CTP:phosphoethanolamine cytidylyltransferase. In these mutants, light-evoked photoreceptor potentials persisted after 20-s light stimulation, indicating prolonged activation of the visual response. These mutants also exhibited light-independent degeneration of photoreceptor neurons, and lipidomic analysis revealed alterations in major phospholipid composition. Phospholipid composition was manipulated via comprehensive genetic interactions and it was concluded that pect mutant phenotypes resulted from PE deficiency. Strikingly, increasing PE synthesis through the PSD pathway effectively suppressed retinal degeneration in pect mutants. Finally, the Mitochondria Associated Membrane (MAM)-enriched proteins MFN and SERCA were required for PSD to rescue pect phenotypes. A model is proposed in which PE synthesized in the mitochondria through PSD is transported back to the ER through endoplasmic reticulum-mitochondria contact sites (ERMCS) when cellular PE is deficient (Zhou, 2020).

Membrane phospholipids, in particular PE, play key roles in regulating neuronal activity and integrity. Drosophila photoreceptor neurons utilize the fastest phospholipid signaling cascade and exhibit high rates of membrane trafficking. Therefore, it is particularly important for neurons to maintain phospholipid pools. As phototransduction is completely mediated by phospholipase C (PLC), maintaining levels of PIP2 and its product DAG are critical for visual responses. Mutations in the gene pect prevent the synthesis of PE from DAG, resulting in increased levels of PI and DAG, and prolonged visual responses. Reducing DAG levels by either introducing mutations in the Lazaro enzyme, which converts PA to DAG, or overexpressing the DAG kinase rdgA, did not suppress the prolonged afterpotentials in pect mutants. Moreover, inhibiting PI synthesis failed to suppress the retinal degeneration and defective ERG responses. In addition, TRP channels were constitutively active in rdgA mutant photoreceptors, and the over-activation and retinal degeneration of rdgA mutants were rescued in rdgA;trp double mutants. In the case of pect, loss of TRP channels did not affect the severity of neurodegeneration, indicating that RDGA and PECT function in different pathways. In conclusion, PECT regulates photoreceptor function and morphology independent of PI and DAG metabolism. Recent evidence suggested TRPs are mechanosensitive channels, and membrane physical properties are involved in channel activation. Indeed, the rhabdomere size was reduced in the 1-day-old pect mutants, indicating changes in the physical properties of the lipid bilayer. It is speculated that alterations in phospholipid composition in pect mutants may change the compact structure and membrane fluidity of rhabdomere, which leads to prolonged activation of TRP channels (Zhou, 2020).

Direct evidence is provided that maintaining cellular PE levels is critical for neuronal function and integrity. First, disruption of the PSD pathway by knocking down PSD enhanced retinal degeneration in pect mutants. More importantly, overexpression of PSD restored cellular PE levels, and thus greatly suppressed both the prolonged afterpotentials in response to light and retinal degeneration of pect mutants. Moreover, this suppression was fully reversed by down-regulating PSS. It has been reported that knockdown of Psd resulted in light-dependent retinal degeneration by preventing autophagy-dependent rhodopsin degradation since the abundance of PE could positively regulate autophagy. However, for pect mutants, rhodopsin turn-over is normal and degeneration is independent of light and TRP channel activity, suggesting that rhodopsin homeostasis does not cause pect-induced cell death. Furthermore, the light-independent retinal degeneration in pect mutants suggested that cellular PE homeostasis independently contributes to maintaining neuronal activity and integrity (Zhou, 2020).

The majority of mitochondrial PE is synthesized in situ in mitochondria via PSD. In contrast, only a small fraction of mitochondrial PE is made in the ER by the CDP-ethanolamine pathway. Maintaining mitochondrial PE levels is critical for mitochondrial respiratory capacity, morphology, and distribution, thus deletion of PSD impairs mitochondrial function, resulting in lethality. The pect mutant photoreceptor cells are dysfunctional, and both cellular and mitochondrial levels of PE are significantly reduced. Although it has been reported that < 30% depletion of mitochondrial PE by RNAi silencing of PSD alters mitochondrial morphology and function in mammalian cells, ~40% reduction in mitochondrial PE levels in pect²⁹ flies did not affect mitochondrial activity, morphology, or axonal localization. Overexpression of PSD, which completely rescued photoreceptor function and integrity in pect mutants, restored total PE levels but not levels of mitochondrial PE. Therefore, disruption of the CDP-ethanolamine PE synthesis pathway did not impair mitochondrial function. Disrupting PE synthesis through the CDP-ethanolamine pathway does not result in a mitochondrial phenotype because mitochondrial PE is synthesized locally through PSD, although the CDP-ethanolamine pathway does contribute to total mitochondrial PE levels (Zhou, 2020).

A recent study reported that yeast Psd1 localizes to both mitochondria and the ER through its transmembrane region (TMR). This forced the authors to consider that PSD in the ER may have restored cellular PE homeostasis in pect mutants. However, Drosophila PSD lacks the TMR sequence required for Psd1 to localize to the ER, and PSD remains in mitochondria when overexpression. ER-mitochondria connections are necessary for the efficient exchange of phospholipids between organelles. Several proteins, including the mitofusin MFN2 and the ER Ca2+ ATPase SERCA, have been implicated in maintaining ERMCS, thus facilitating phospholipid exchange. Consistent with previous reports, this study found that the fly proteins, MFN and SERCA, stabilize ER-mitochondrial contact sites. Importantly, disrupting ER-mitochondria contacts through mfnRNAi or sercaRNAi completely blocked the ability of PSD to rescue pect mutant phenotypes. This is also consistent with a recent study that described an unexpected role of MFN2 in PS transfer between the ER and mitochondria. However, as loss of SERCA induces UPR reaction, the possible role of inducing UPR in disruption of PE homeostasis cannot be ruled out. Taken together, these analyses prove that cellular PE homeostasis is maintained, in part, by synthesizing PE in mitochondria and then exporting this PE to cellular pools. Thus, mitochondria play a critical role in cellular phospholipid homeostasis (Zhou, 2020).

PE species generated from the CDP-ethanolamine and PSD pathways are different, especially fatty acids in the sn-2 position, where PE from the CDP-ethanolamine and PSD pathways prefer mono-/di-unsaturated and polyunsaturated fatty acids, respectively. It has been suggested that individual molecular species of PE may play specialized roles in cellular signaling, which explained why deletion of either Pisd or Pcyt2 causes embryonic lethality in mice. This study saw that as total PE levels decreased, the proportion of most PE species were unaffected. In contrast, the PE species PE38:1, PE36:4, and PE36:5 were down-regulated, and PSD overexpression fully restored levels of these three PE species, as well as total PE levels. This suggests that the same PE species are generated by the CDP-ethanolamine and PSD pathway in vivo, although they might prefer different substrates in vitro. These data further showed that PE generated in the mitochondria can compensate for cellular PE deficiency in the Drosophila visual system (Zhou, 2020).

A recent study also identified recessive lethal mutations in pect and demonstrated that the biosynthesis of specific phospholipids is linked to neurodegeneration and synaptic vesicle loss in adult Drosophila photoreceptors (Tsai, 2019). In pect^omb593 mutants levels of PE 34:1 and PE 36:2 were reduced, but the overall proportions of PE species were not significantly changed, whereas this study detected a significant reduction in total cellular PE levels. The different results of phospholipid composition in pect mutants may come from the different alleles analyzed. In contrast to the nonsense pect²⁹ mutation, the pect^omb593 contains a hypomorphic mutation with a single amino acid change (H55Y). Thus PECTH55Y might reduce PE levels to a much lesser extent than complete loss of pect, and this cellular PE reduction could be compensated by the alternative PSD pathway. Moreover, knocking down >srebp (regulatory element binding protein) reversed the loss of synaptic vesicles, but failed to suppress axonal or retinal degeneration in pect^omb593 mutants. Therefore, SREBP is a downstream factor of PE deficiency and specifically functions in maintaining the synaptic vesicle pools. This study demonstrated that increasing PE levels through induction of the PSD pathway restored the cellular PE levels and effectively suppressed retinal degeneration and defective phototransduction in pect mutants, supporting disruption of PE homeostasis is the major cause of photoreceptor cell degeneration and loss of synaptic transmission (Zhou, 2020).

Phospholipid composition is critical for cellular homeostasis, and alterations in the composition of major phospholipid PE are implicated in multiple diseases. Cellular PC/PE molar ratios can influence energy metabolism in numerous organelles and thus lead to disease conditions such as steatohepatitis, obesity, and muscular dystrophy. Cellular PC levels were elevated by 57% in pect²⁹ retinas compared with wild type, but inhibiting PC synthesis did not suppress the retinal degeneration, suggesting that PE levels, but not the PC/PE ratio, are crucial for neuronal function (Zhou, 2020).

Genetic mutations that affect PE synthesis have been identified in several human autosomal-recessive disorders. In particular, mutations in PISD, the human counterpart of fly PSD, cause Liberfarb syndrome, which is a multisystem disorder affecting the eyes, ears, bone, and brain. Studies in patient-derived fibroblasts revealed impaired phospholipid metabolism, altered mitochondrial respiration, and fragmentation of the mitochondrial network. Recently, mutations in PCYT2, the human counterpart of fly PECT, have been associated with hereditary spastic paraplegia. Lipidomic analysis of patient fibroblasts revealed profound lipid abnormalities impacting both neutral etherlipid and etherphospholipid metabolism. As mutations in fly pect affect phospholipid composition, resulting in defective photoresponse and severe retinal degeneration, this system represents a conserved model for studying diseases associated with PE deficiency. Moreover, these data provide direct genetic evidence that induction of mitochondrial PE synthesis can compensate for deficiencies in cellular PE, and suggest that mitochondrial phospholipid synthesis and trafficking represent a promising therapeutic target for treating disorders associated with defective phospholipid composition (Zhou, 2020).

Transcriptional Feedback Links Lipid Synthesis to Synaptic Vesicle Pools in Drosophila Photoreceptors

Neurons can maintain stable synaptic connections across adult life. However, the signals that regulate expression of synaptic proteins in the mature brain are incompletely understood. This study describes a transcriptional feedback loop between the biosynthesis and repertoire of specific phospholipids and the synaptic vesicle pool in adult Drosophila photoreceptors. Mutations that disrupt biosynthesis of a subset of phospholipids cause degeneration of the axon terminal and loss of synaptic vesicles. Although degeneration of the axon terminal is dependent on neural activity, activation of sterol regulatory element binding protein (SREBP) is both necessary and sufficient to cause synaptic vesicle loss. These studies demonstrate that SREBP regulates synaptic vesicle levels by interacting with tetraspanins, critical organizers of membranous organelles. SREBP is an evolutionarily conserved regulator of lipid biosynthesis in non-neuronal cells; these studies reveal a surprising role for this feedback loop in maintaining synaptic vesicle pools in the adult brain (Tsai, 2019).

These studies demonstrate that disrupting the biosynthesis of specific membrane phospholipids causes adult-onset degeneration of R cells and loss of synaptic vesicles. These two phenotypes arise via distinct molecular mechanisms that can be doubly dissociated using genetic and physiological manipulations. Degeneration of the axon terminal is an activity-dependent process that requires calcium-mediated vesicle fusion. Conversely, loss of synaptic vesicles is driven by activation of the transcription factor SREBP. Thus, in these cells, SREBP is activated by alterations in the levels of specific phospholipids. Here, SREBP affects the expression of a specific subset of genes that are largely not directly involved in lipid regulation, thus defining a previously unknown SREBP function. Rather, SREBP activation leads to reduced expression of four tetraspanins. Restoring expression of either of two of these tetraspanins suppresses the effects of SREBP activation, demonstrating that tetraspanins are functional effectors of SREBP in photoreceptors. Thus, a specialized feedback loop from the synaptic terminal to the nucleus links the levels of specific phospholipids to photoreceptor function and synaptic vesicle number. It is proposed that this feedback loop matches the vesicular demand for phospholipids to their production. As SREBP is evolutionarily conserved, and recent studies have linked SREBP to neuronal damage in several contexts, it is speculated that this feedback loop plays a central role in maintaining synaptic vesicle pools in the healthy aging brain (Tsai, 2019).

In Drosophila, mutations that disrupt phospholipid biosynthesis cause broad defects in brain function, including increased seizure activity and photoreceptor degeneration. However, these and other studies examining phospholipid composition in flies have either not quantified phospholipid levels or have not differentiated different phospholipid species. These studies using a high-resolution lipidomic approach demonstrate that biosynthesis of specific PE and PC species is required for maintaining synaptic vesicle pools and the axon terminal in adult photoreceptors. Moreover, the biosynthetic enzyme Pect is found at the axon terminal. It is speculated that the production of specific phospholipids can occur locally, coupling precise levels of phospholipids to the cellular processes that require them in the axonal compartment. Finally, recent work has demonstrated that derivatives of very long chain (VLC) PC species are neuroprotective in vertebrate photoreceptors and neurons. Although this study detected only one VLC PC precursor, PC c44:12, representing 0.01% of the total PC species in the fly retina, future studies will determine the extent to which derivatives of this or other PC or PE species play roles in maintaining adult photoreceptor axons and synapses in Drosophila. This work suggests the following model. The ultrastructural analysis of pect mutants reveals phenotypes in the axon terminal that are strongly reminiscent of those in endocytic mutants. Consistent with this, blocking exocytosis in pect mutants by either reducing light exposure or by genetic means suppresses axon terminal degeneration. It is therefore inferred that the inability to retrieve synaptic vesicles from the plasma membrane is sufficient to cause neuronal degeneration and that the availability of specific phospholipids can be rate limiting for endocytosis. These results are consistent with previous studies in C. elegans that demonstrated that a phospholipid desaturase causes defects in endocytosis through effects on synaptojanin, a critical component in endocytosis. At the same time, altering phospholipid production may also impair vesicle biogenesis, in which case blocking synaptic transmission could suppress neuronal degeneration by removing the demand for vesicle biogenesis via an as-yet-unknown mechanism (Tsai, 2019).

SREBP is a central regulator of genes involved in lipid biosynthesis in many cell types. The current data support the notion that SREBP plays an additional role in Drosophila photoreceptors. As the levels of only a few phospholipids are altered in pect mutants, SREBP activation appears linked to the detection of changes in levels in these PE and PC species. Moreover, although activation of SREBP does upregulate a small number of genes involved in lipid biosynthesis, it also downregulates many genes involved in phototransduction and synaptic function. Among these, genetic interaction studies demonstrate that tetraspanins are functionally critical SREBP effectors. Tetraspanins are transmembrane proteins that have been linked to synapse development, lysosomal function in R cells, and to outer segment structure and function in the vertebrate retina. Moreover, recent work has demonstrated that they can serve as cholesterol-binding proteins, further implicating this family in the regulation of membrane function. Although unraveling the specific molecular mechanisms that link tetraspanin function to synaptic vesicle pools remains a challenge for future studies, the current model for this role of SREBP represents an extension of SREBP's long-standing role in regulating lipid biosynthesis. In particular, a central role for phospholipids that is unique to neurons is as a critical component of synaptic vesicles. It is hypothesized that, when SREBP is activated and tetraspanin expression is reduced, either the biogenesis of synaptic vesicles is downregulated or their turnover and degradation is increased, shrinking the synaptic vesicle pool in an activity-independent manner. As a result, the cellular demand for the specific phospholipids found in synaptic vesicles is reduced. More broadly, these studies suggest that SREBP might complement its long-standing role in lipid biosynthesis with an additional role in controlling phospholipid utilization. Finally, by combining the high-resolution lipidomic approach this study developed to work with small populations of labeled cells with the powerful genetic tools available in this system, future work may shed further light on the regulation of SREBP activity and phospholipid levels (Tsai, 2019).

SREBP has been linked to both neurodegenerative disease and stroke. Recent studies in flies have demonstrated that reactive oxygen species can activate SREBP to cause lipid droplet formation in glia. However, the molecular mechanisms by which SREBP might act in these contexts are unknown. In addition, mutations in a human tetraspanins have been linked to intellectual disability. The demonstration that SREBP acts through tetraspanins to regulate synaptic vesicle pools and negatively regulates other genes required for synaptic function suggests a unifying mechanism for these seemingly disparate observations. Taken together, these studies argue that SREBP plays an evolutionarily conserved role in regulating neuronal and synaptic function, suggesting a link between the neuronal phospholipid repertoire and synapse maintenance in the adult brain (Tsai, 2019).

Phospholipid homeostasis regulates lipid metabolism and cardiac function through SREBP signaling in Drosophila

The epidemic of obesity and diabetes is causing an increased incidence of dyslipidemia-related heart failure. While the primary etiology of lipotoxic cardiomyopathy is an elevation of lipid levels resulting from an imbalance in energy availability and expenditure, increasing evidence suggests a relationship between dysregulation of membrane phospholipid homeostasis and lipid-induced cardiomyopathy. The present study reports that the Drosophila easily shocked (eas) mutants that harbor a disturbance in phosphatidylethanolamine (PE) synthesis display tachycardia and defects in cardiac relaxation and are prone to developing cardiac arrest and fibrillation under stress. Phosphatidylethanolamine (PE) is the second most abundant phospholipid in mammals and the major phospholipid in most dipterans. The CDP-ethanolamine pathway is the principal route for PE synthesis in most mammalian tissues, with the first step requiring ethanolamine kinase. In Drosophila, the easily shocked (eas)-encoded ethanolamine kinase catalyzes the phosphorylation of ethanolamine to phosphoethanolamine. Phosphoethanolamine is further modified by phosphoethanolamine cytidylytransferase (PECT) to produce CDP-ethanolamine, which, together with diacylglycerol (DAG), generates PE. eas mutant hearts exhibit elevated concentrations of triglycerides, suggestive of a metabolic, diabetic-like heart phenotype. Moreover, the low PE levels in eas flies mimic the effects of cholesterol deficiency in vertebrates by stimulating the Drosophila sterol regulatory element-binding protein (dSREBP) pathway. Significantly, cardiac-specific elevation of dSREBP signaling adversely affects heart function, reflecting the cardiac eas phenotype, whereas suppressing dSREBP or lipogenic target gene function in eas hearts rescues the cardiac hyperlipidemia and heart function disorders. These findings suggest that dysregulated phospholipid signaling that alters SREBP activity contributes to the progression of impaired heart function in flies and identifies a potential link to lipotoxic cardiac diseases in humans (Lim, 2011).

This study used Drosophila genetic approaches to identify a novel metabolic cardiomyopathy that exhibits striking features of obesity- and diabetes-related heart failure in humans. Specifically, it was shown that a genetically dysregulated phospholipid metabolism leads to chronic stimulation of the transcription factor dSREBP and its lipogenic target genes, which in turn leads to cardiac fat accumulation associated with electrical and functional signatures of heart failure. This study highlights a regulatory relationship between the PE phospholipid and TG metabolism that could play a major role in eliciting cardiac steatosis and dysfunction, and identifies the dSREBP signaling pathway as the key metabolic pathway that underlies the increased synthesis and accumulation of TG upon the disruption of PE homeostasis (Lim, 2011).

The current data lead to a model that describes how the dysregulation of membrane PE homeostasis could promote the pathogenesis of lipotoxic cardiomyopathy. In wild-type flies, a decrease in membrane PE level triggers the proteolytic release of a transcriptionally active form of dSREBP (m-dSREBP) and induces the biosynthesis of fatty acids in a manner similar to that in mammals. Upon the subsequent use of these fatty acids in PE synthesis, and the restoration of normal PE concentrations in cellular membranes, further processing of dSREBP is blocked and overall lipid synthesis is reduced. The presence of such a feedback inhibitory loop ensures that PE homeostasis can be achieved under physiological conditions. In flies harboring a genetic perturbation of the CDP-ethanolamine pathway, the failure to produce PE and the ensuing low levels of PE disrupt the homeostatic negative feedback loop, resulting in the continuous activation of the dSREBP pathway. Prolonged stimulation of lipogenesis and the oversupply of lipid intermediates such as acyl coA and DAG could lead to increased production of TG, resulting in hypertriglyceridemia, cardiac steatosis, and the progressive development of lipotoxic cardiomyopathy (Lim, 2011).

It is possible that the above phenomenon, although identified in a fly model, also occurs in mammals. In fact, in mice, elimination of the CDP-ethanolamine pathway resulting in the absence of PE synthesis induced a significant elevation of TG levels. Along with hypertriglyceridemia, it was also observed in these studies that the expression of key fatty acid biosynthetic genes such as ACC and FAS is up-regulated in PE-deficient mice. It has been proposed that the elevated TG concentration is due to an increased availability of DAG arising from its underutilization by the CDP-ethanolamine pathway that leads to a redirection of DAG to TG formation. However, this proposal fails to explain how the passive accumulation of DAG in the PE-deprived state could induce an upstream event such as the expression of the lipogenic genes. The mechanism proposed in this model based on the eas² fly studies could reconcile to some extent this dilemma in the mammalian system. The model posits that constitutively low levels of PE drive a compensatory hyperactivation of the SREBP pathway. Once activated, SREBP can induce de novo lipogenesis and the active generation of intermediates such as acyl coA and DAG, a sequence of steps that culminates in the heightened production of TG. Indeed, in mice lacking the capacity to generate PE, the expression of one of the mammalian SREBP isoforms, SREBP-1c, was found to be up-regulated. Furthermore, the PE-deficient mice also develop metabolic disorders such as hepatic steatosis and insulin resistance. However, it remains to be seen whether SREBP signaling might similarly be regulated by PE homeostasis in mammals such that a deficit in PE levels elicits an activation of the SREBP pathway to generate increased amounts of fatty acids and DAG/TG. It would be interesting to test whether the enhanced levels of TGs, as well as the severity of these phenotypes, would be significantly ameliorated upon the down-regulation of SREBP expression or activity in these mice, indicating a primary role of SREBP signaling in mediating the development of hypertriglyceridemia and its related metabolic disorders upon the perturbation of PE synthesis in the mammalian context (Lim, 2011).

This model, based on studies in Drosophila eas mutants, provides insights into the potential role of the dSREBP signaling pathway in coupling membrane phospholipid homeostasis with lipid metabolism and its associated metabolic functions. These findings also support the notion that Drosophila shares many of the basic metabolic functions found in vertebrates, and that the genetic dissection of the metabolic and transcriptional responses in a less complex model organism such as Drosophila facilitates understanding of fundamental aspects of metabolic control, cardiac physiology, and associated disease mechanisms (Lim, 2011).

Skeletal Muscle Consequences of Phosphatidylethanolamine Synthesis Deficiency

The maintenance of phospholipid homeostasis is increasingly being implicated in metabolic health. Phosphatidylethanolamine (PE) is the most abundant phospholipid on the inner leaflet of cellular membranes, and previous work has shown that mice with a heterozygous ablation of the PE synthesizing enzyme, Pcyt2 (Pcyt2(+/-)), develop obesity, insulin resistance, and non-alcoholic steatohepatitis (NASH). Skeletal muscle is a major determinant of systemic energy metabolism, making it a key player in metabolic disease development. Both the total PE levels and the ratio of PE to other membrane lipids in skeletal muscle are implicated in insulin resistance; however, the underlying mechanisms and the role of Pcyt2 regulation in this association remain unclear. This study shows how reduced phospholipid synthesis due to Pcyt2 deficiency causes Pcyt2(+/-) skeletal muscle dysfunction and metabolic abnormalities. Pcyt2(+/-) skeletal muscle exhibits damage and degeneration, with skeletal muscle cell vacuolization, disordered sarcomeres, mitochondria ultrastructure irregularities and paucity, inflammation, and fibrosis. There is intramuscular adipose tissue accumulation, and major disturbances in lipid metabolism with impaired FA mobilization and oxidation, elevated lipogenesis, and long-chain fatty acyl-CoA, diacylglycerol, and triacylglycerol accumulation. Pcyt2(+/-) skeletal muscle exhibits perturbed glucose metabolism with elevated glycogen content, impaired insulin signaling, and reduced glucose uptake. Together, this study lends insight into the critical role of PE homeostasis in skeletal muscle metabolism and health with broad implications on metabolic disease development (Grapentine, 2023).

PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. This study shows that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing (Cikes, 2023).

The development of a metabolic disease phenotype in CTP:phosphoethanolamine cytidylyltransferase-deficient mice

Phosphatidylethanolamine (PE) is an important inner membrane phospholipid mostly synthesized de novo via the PE-Kennedy pathway and by the decarboxylation of phosphatidylserine. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the formation of CDP-ethanolamine, which is often the rate regulatory step in the PE-Kennedy pathway. The current investigation shows that the reduced CDP-ethanolamine formation in Pcyt2(+/-) mice limits the rate of PE synthesis and increases the availability of diacylglycerol. This results in the increased formation of triglycerides, which is facilitated by stimulated de novo fatty acid synthesis and increased uptake of pre-existing fatty acids. Pcyt2(+/-) mice progressively accumulate more diacylglycerol and triglycerides with age and have modified fatty acid composition, predominantly in PE and triglycerides. Pcyt2(+/-) additionally have an inherent blockage in fatty acid utilization as energy substrate and develop impaired tolerance to glucose and insulin at an older age. Accordingly, gene expression analyses demonstrated the up-regulation of the main lipogenic genes and down-regulation of mitochondrial fatty acid beta-oxidation genes. These data demonstrate for the first time that to preserve membrane PE phospholipids, Pcyt2 deficiency generates compensatory changes in triglyceride and energy substrate metabolism, resulting in a progressive development of liver steatosis, hypertriglyceridemia, obesity, and insulin resistance, the main features of the metabolic syndrome (Fullerton, 2009).

Search PubMed for articles about Drosophila Pect

Cikes, D., Elsayad, K., Sezgin, E., Koitai, E., Torma, F., Orthofer, M., Yarwood, R., Heinz, L. X., Sedlyarov, V., Miranda, N. D., Taylor, A., Grapentine, S., Al-Murshedi, F., Abot, A., Weidinger, A., Kutchukian, C., Sanchez, C., Cronin, S. J. F., Novatchkova, M., Kavirayani, A., Schuetz, T., Haubner, B., Haas, L., Hagelkruys, A., Jackowski, S., Kozlov, A. V., Jacquemond, V., Knauf, C., Superti-Furga, G., Rullman, E., Gustafsson, T., McDermot, J., Lowe, M., Radak, Z., Chamberlain, J. S., Bakovic, M., Banka, S., Penninger, J. M. (2023). PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing. Nat Metab, 5(3):495-515 PubMed ID: 36941451

de Wit, N. J., Bosch-Vermeulen, H., de Groot, P. J., Hooiveld, G. J., Bromhaar, M. M., Jansen, J., Muller, M., van der Meer, R. (2008). The role of the small intestine in the development of dietary fat-induced obesity and insulin resistance in C57BL/6J mice. BMC Med Genomics, 1:14 PubMed ID: 18457598

Dobrosotskaya, I. Y., Seegmiller, A. C., Brown, M. S., Goldstein, J. L., Rawson, R. B. (2002). Regulation of SREBP processing and membrane lipid production by phospholipids in Drosophila. Science, 296(5569):879-883 PubMed ID: 11988566

Fullerton, M. D., Hakimuddin, F., Bonen, A., Bakovic, M. (2009). The development of a metabolic disease phenotype in CTP:phosphoethanolamine cytidylyltransferase-deficient mice. J Biol Chem, 284(38):25704-25713 PubMed ID: 19625253

Grapentine, S., Singh, R. K., Bakovic, M. (2023). Skeletal Muscle Consequences of Phosphatidylethanolamine Synthesis Deficiency. Function (Oxf), 4(4):zqad020 PubMed ID: 37342414

Kelly, K. P., Alassaf, M., Sullivan, C. E., Brent, A. E., Goldberg, Z. H., Poling, M. E., Dubrulle, J. and Rajan, A. (2022). Fat body phospholipid state dictates hunger-driven feeding behavior. Elife 11. PubMed ID: 36201241

Lim, H. Y., Wang, W., Wessells, R. J., Ocorr, K., Bodmer, R. (2011). Phospholipid homeostasis regulates lipid metabolism and cardiac function through SREBP signaling in Drosophila. Genes Dev, 25(2):189-200 PubMed ID: 21245170

Tsai, J. W., Kostyleva, R., Chen, P. L., Rivas-Serna, I. M., Clandinin, M. T., Meinertzhagen, I. A., Clandinin, T. R. (2019). Transcriptional Feedback Links Lipid Synthesis to Synaptic Vesicle Pools in Drosophila Photoreceptors. Neuron, 101(4):721-737 e724 PubMed ID: 30737130

Zhao, H. and Wang, T. (2020). PE homeostasis rebalanced through mitochondria-ER lipid exchange prevents retinal degeneration in Drosophila. PLoS Genet 16(10): e1009070. PubMed ID: 33064773

date revised: 26 February 2024

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