Intrinsic and extrinsic regulation of rhabdomyolysis susceptibility by Tango2

ABSTRACT Rhabdomyolysis is a clinical emergency characterized by severe muscle damage, resulting in the release of intracellular muscle components, which leads to myoglobinuria and, in severe cases, acute kidney failure. Rhabdomyolysis is caused by genetic factors linked to increased disease susceptibility in response to extrinsic triggers. Recessive mutations in TANGO2 result in episodic rhabdomyolysis, metabolic crises, encephalopathy and cardiac arrhythmia. The underlying mechanism contributing to disease onset in response to specific triggers remains unclear. To address these challenges, we created a zebrafish model of Tango2 deficiency. Here, we demonstrate that the loss of Tango2 in zebrafish results in growth defects, early lethality and increased susceptibility of skeletal muscle defects in response to extrinsic triggers, similar to TANGO2-deficient patients. Using lipidomics, we identified alterations in the glycerolipid pathway in tango2 mutants, which is critical for membrane stability and energy balance. Therefore, these studies provide insight into key disease processes in Tango2 deficiency and have increased our understanding of the impacts of specific defects on predisposition to environmental triggers in TANGO2-related disorders.


Introduction
Rhabdomyolysis (RM) is a complex medical disorder involving catastrophic failure of skeletal muscle homeostasis and integrity, resulting in muscle breakdown and release of muscle cytosolic content into the circulation (Cabrera-Serrano and Ravenscroft, 2022). Rhabdomyolysis can give rise to serious health complications such as myoglobinuria, cardiac arrhythmia, and acute kidney injury. Clinical symptoms can include severe muscle weakness, myalgia, and muscle swelling, with a serum creatine kinase (CK) rising above 1000U/l. Rare disease-causing mutations are associated with a small but significant subset (~15%) of rhabdomyolysis patients.
Environmental factors such as viral infections (SARS-CoV-2, HIV), physical exertion, and certain medications are major contributing factors in combination with a genetic predisposition (Szugye, 2020, East et al., 1988, Rawson et al., 2017, Wu et al., 2022, Knoblauch et al., 2013. Even in genetic forms of rhabdomyolysis, environmental factors increase the susceptibility to recurrent episodes of muscle breakdown . Environmental factors contributing to rhabdomyolysis have been mostly identified through life-threatening reactions to different triggers in the clinical population. Lack of clear understanding of intrinsic disease mechanism also prevents investigation of the role that extrinsic factors have on increasing the susceptibility of muscle damage in predisposing genotypes. Recessive mutations in TANGO2 underlie a rare pediatric disorder resulting in encephalopathy, rhabdomyolysis, and cardiac abnormalities (Lalani et al., 2016, Kremer et al., 2016, Miyake et al., 2022. TANGO2 encodes for "Transport and Golgi Organization 2" proteins, first identified in a genetic screen for proteins required in constitutive protein secretion in Drosophila cells (Bard et al., 2006). Depletion of Tango2 results in the fusion of endoplasmic reticulum and Golgi compartments in Drosophila cells. The fibroblasts of TANGO2 deficient patients exhibit a profound decrease in the endoplasmic reticulum area (Lalani et al., 2016). Functional studies in patient fibroblasts have shown that TANGO2 is required for ER-Golgi trafficking in cells (Milev et al., 2021). Proteomic analysis of fibroblasts from TANGO2 patients revealed significant changes in the component of mitochondrial fatty acid oxidation, plasma Disease Models & Mechanisms • DMM • Accepted manuscript membrane, endoplasmic reticulum, Golgi, and secretory pathway indicating pleiotropic roles in the disease biology (Mingirulli et al., 2020). Some patients' fibroblasts also showed a defect in palmitate-dependent oxygen consumption, suggesting an impaired mitochondrial fatty acid oxidation (Kremer et al., 2016, Heiman et al., 2022. In contrast, myoblasts from TANGO2 patients exhibit no defects in mitochondrial structure and function but exhibit abnormalities in mitochondrial function under nutrient stress. This suggests that TANGO2 may have different functions in different cell types in response to extrinsic triggers (Bérat et al., 2021). These studies indicate that TANGO2 deficiency results in intrinsic metabolic defects exacerbated under stress conditions. However, a clear understanding of the disease processes resulting in the pathological state is still lacking.
TANGO2 mutations result in clinical heterogeneity in affected patients. Muscle weakness and neurodevelopmental presentation often precede life-threatening complications of rhabdomyolysis, cardiac arrhythmias, or cardiomyopathy. However, a clear genotype-phenotype correlation is lacking in these patients (Powell et al., 2021).
The presence of variable phenotypes in different cell types suggests that TANGO2 may play diverse roles in various cell types in vivo. Tango2 knockout mice exhibit normal development, lifespan, and physiology (International Mouse Phenotyping Consortium). Therefore, robust model systems are needed to understand the effect of TANGO2 deficiency on variable clinical presentation and to identify the pathological processes contributing to disease onset and progression. To address these challenges, we developed vertebrate zebrafish models of Tango2 deficiency. Tango2 deficiency results in normal embryonic development in zebrafish but causes increased lethality during larval and juvenile stages. The tango2 mutant larval zebrafish develops acute muscle dysfunction by extrinsic stress triggers. Global lipidomics identified a reduced abundance of lipids synthesized by ER/SR localized Glycerol-3-phosphate pathway enzymes in Tango2 deficiency which are critical for cellular membranes and energy states. The studies presented in this work provide mechanistic insights into intrinsic disease processes and extrinsic risk factors for increased susceptibility to RM in TANGO2-related disorders.

Tango2 deficiency in zebrafish results in normal embryonic development but increased lethality during larval and juvenile stages
Zebrafish grow ex vivo, so disease onset and progression can be visualized in individual animals. To understand the role of Tango2 in vivo in disease onset and progression, we created a loss of function tango2 alleles in zebrafish using the CRISPR-Cas9 system. The tango2 gene in zebrafish encodes for two transcripts, and therefore sgRNAs were designed to knock out both transcripts ( Figure 1A). tango2 alleles created include tango2 bwg210 with insertion of seven bases (c.226_227ins7; p.Tyr76Leufs*25) and tango2 bwg211 with insertion of 26 bases in exon2 (c.226_227ins26; p.Tyr76Leufs*207) ( Figure 1B). These alleles result in out-of-frame mutations and are predicted to result in truncating proteins. Phenotypic analysis of tango2 bwg210 and tango2 bwg211 lines revealed no significant differences. Therefore, the tango2 bwg211 line (referred to as tango2 mutants) was used for further investigation unless specified. To validate the effect of c.226_227ins26 mutation at the protein level, Western blot analysis was performed on control and tango2 zebrafish (4 weeks) and showed a complete absence of Tango2 protein in the mutant fish ( Figure 1C and Figure S1). Therefore c.226_227ins26 mutation results in the loss of function of the Tango2 protein in zebrafish. Phenotypic analysis of control and tango2 mutant larval zebrafish showed no significant morphological differences during early development ( Figure 1D, 8 days post fertilization; dpf). To identify any effect of Tango2 deficiency on the lifespan of mutant fish, control (+/+ HT ) and (-/-HT ) genotypes obtained from heterozygous parents were analyzed until 3 months of age. Despite no obvious morphological differences in control and mutant larval fish at early stages, a reduced survival rate was observed for mutant fish from 7 days post fertilization, and 96% of the mutant fish died by 3 months of age ( Figure 1E). tango2 mutants (-/-M ) obtained from tango2 parents also showed similar lifespans compared to tango2 mutants (-/-HT ) obtained from heterozygous parents suggesting a lack of maternal tango2 mRNA effect, reported previously for another allele of tango2 (Sun et al., 2022). This was further validated by the absence of tango2 mRNA in tango2 mutants (-/-HT ) ( Figure S1).
Therefore, control and mutants obtained from the heterozygous tango2 bwg211 line were Disease Models & Mechanisms • DMM • Accepted manuscript used in subsequent studies. Quantifying body weight also showed a significant reduction in weights of tango2 mutants compared to wild-type siblings at four weeks of age, indicative of growth defects ( Figure 1F). Histological analysis of skeletal muscle revealed a smaller myofiber size than controls ( Figure S2). Together, these studies suggest that Tango2 deficiency in zebrafish results in muscle growth defects and early lethality in larval and juvenile stages.

Tango2 is localized at endo-membranes in myofibers.
TANGO2 mutations result in a decreased network of the endoplasmic reticulum in patient fibroblasts and metabolic abnormalities indicative of the involvement of intracellular organelles in disease pathology. To address the localization of Tango2 skeletal muscle, immunofluorescence was performed on myofibers isolated from zebrafish (45 days). Co-localization with different skeletal muscle proteins showed colocalization of Tango2 with Ryr1 (sarcoplasmic reticulum protein), and Golga2 (Golgi apparatus) ( Figure 2A) in proximity to the mitochondria (Tomm20). Tango2 was previously identified in a genomic screen on the regulators of ER-Golgi trafficking.
Therefore, the co-localization of Tango2 with the endomembrane compartments suggests potential roles in the maintenance or function of these organelles in the skeletal muscle (Bard et al., 2006). To understand the effect of Tango2 deficiency on these membrane compartments in skeletal muscle, immunofluorescence was performed on control and tango2 mutant myofibers. Mutant myofibers displayed local regions of disorganized Ryr1 immunofluorescence, indicating structural defects in SR in these areas ( Figure 2B-C, arrow). Quantification of the myofibers with regions of disorganized Ryr1 staining showed an increase in these regions in the tango2 mutant zebrafish ( Figure 2C). Moreover, reduced or no mitochondrial staining regions were also observed in the mutant myofibers ( Figure 2B-C, arrow). Quantification of these regions showed an increase in tango2 myofibers compared to controls ( Figure 2C).
No differences in the Golgi apparatus were identified between control and tango2 mutant myofibers. Therefore, Tango2 is localized and required to maintain the endomembrane compartments' structural organization.

Disease Models & Mechanisms • DMM • Accepted manuscript
tango2 mutant fish exhibit ultrastructural defects in the sarcoplasmic reticulum and mitochondria.
To identify the temporal changes of structural abnormalities observed in myofibers in larval fish (60 days, Figure 2), skeletal muscle ultrastructure was examined during early larval development (8 dpf) when control and mutant fish are phenotypically and functionally similar. Ultrastructural evaluation of sarcomeres by electron microscopy showed no significant defects in tango mutants' sarcomere size (length or height) compared to control siblings ( Figure 3A-B). While most of the sarcoplasmic reticulum and mitochondria were normal in tango2 mutants, 10-12%, myofibers exhibited defects in both sarcoplasmic reticula (13.8  4.9% in mutants Vs. 4.1  0.85% in control, p<0.01) and mitochondria structures (9.2  2.3% in mutants Vs. 0.8%  0.79 in control, p<0.01) ( Figure 3). Defective sarcoplasmic reticulum showed either collapsed or smaller terminal cisternae of sarcoplasmic reticulum in tango2 mutants compared to controls ( Figure 3B-D, arrows). In contrast to longer mitochondria in control muscles, tango2 mutant muscle exhibited smaller mitochondria associated with whorled membrane structures also seen in mitochondrial myopathies ( Figure 3E-H, M) (Vincent et al., 2016). Interestingly, these abnormalities were present together in these affected myofibers, and no myofibers with either sarcoplasmic reticulum or mitochondrial defects were observed. Moreover, an abnormal accumulation of vesicles was also observed in the proximity of the sarcoplasmic reticulum and mitochondria that exhibited similar electron-dense membranes as whorled membrane structures in the mitochondria in the mutant myofibers and were likely derived from damaged mitochondria. These data show that the absence of Tango2 results in ultrastructure defects in the sarcoplasmic reticulum and mitochondria.

SR stress results in skeletal muscle defects in tango2 mutants
tango2 mutant larvae exhibit normal motor function during early larval stages despite mild ultrastructural changes in a small group of myofibers (5-7 dpf, Figure 4). This is similar to many TANGO2 patients that exhibit normal motor function except for the development of acute muscle damage during rhabdomyolysis episodes. tango2 mutant fish exhibit smaller or deflated terminal cisternae of the sarcoplasmic reticulum Disease Models & Mechanisms • DMM • Accepted manuscript occupied by Ryr1 channels (Figure 3). SR is the regulator of excitation-contraction coupling in skeletal muscle through the release of Ca 2+ by the Ryr1 channel and the reuptake of Ca 2+ by the Serca channel (Lawal et al., 2020). Caffeine is an activator of Ryr1 that binds to Ryr1 and increases Ca 2+ sensitivity. To test if Tango2 deficiency increases susceptibility to muscle damage caused by sarcoplasmic reticulum stress, we treated control and tango2 mutant larval fish (6 dpf) with caffeine. We quantified the swimming behavior by using an automated movement-tracking system. Acute caffeine exposure resulted in reduced swimming of controls and tango2 mutant fish. However, the effect was more severe in tango2 mutants than in control siblings ( Figure 4A-B).
While control fish recovered completely after 24 hours of caffeine exposure, tango2 mutants failed to revert to the normal level of motor function. Dantrolene is a Ryr1 antagonist and protects against hypersensitivity of calcium release from the sarcoplasmic reticulum.
Dantrolene is used clinically to control malignant hyperthermia and rhabdomyolysis.
Therefore, we tested the effectiveness of dantrolene in improving acute muscle dysfunction in tango2 mutants induced by caffeine exposure. Control and tango2 mutant larval zebrafish (6 dpf) were treated with caffeine (C) or caffeine and dantrolene (D), and normalized motor function (to pre-caffeine treatment) was analyzed. Caffeine resulted in highly reduced motor function in tango2 larval zebrafish compared to controls ( Figure 4C). Treatment with dantrolene resulted in a small and significant improvement in muscle function in tango2 mutants. Whole mount phalloidin staining of the myotome revealed disorganized myotome in tango2 mutants at the basal state, with several myofibers lacking the parallel myofibers organization seen in the control ( Figure 4D, arrowheads). While no significant myofiber breakdown or atrophy was observed on caffeine exposure, tango2 mutants showed disarray of sarcomere banding pattern with the widening of A-bands between adjacent I-bands (area between phalloidin stained sarcomeres) ( Figure 4D, arrows) that were rescued on dantrolene treatment. Although improved motor function and skeletal muscle structure were observed in tango2 mutants, dantrolene treatment did not result in a complete rescue of muscle structure and function to normal levels.

Exercise-induced skeletal muscle damage in Tango2 deficiency
TANGO2 deficiency in patients is associated with rhabdomyolysis. However, stress conditions leading to rhabdomyolysis in these patients remain mostly unknown.
Exercise-induced rhabdomyolysis is the most common trigger of muscle damage in susceptible individuals in other genetic forms of rhabdomyolysis. Mechanical loading of the skeletal muscle in control and tango2 mutants (8 dpf Tango2 deficiency. Quantifying the phosphatidylcholine that showed the highest reduction in tango2 mutants at early larval stages showed a similar reduction in mutants ( Figure S3A). This suggests that most lipid defects observed in mutants are also present during early larval stages and are not a downstream secondary effect of disease progression. ER/SR harbors enzymes for the glycerol-3-phosphate pathway that synthesizes phospholipids which are major building blocks for lipids in the cellular membrane. Quantification of the glycerol-3-phosphate pathway enzymes that catalyze Disease Models & Mechanisms • DMM • Accepted manuscript lysophosphatidic acid to triacyl glycerol and phospholipids revealed a significant downregulation of agpat1, lpin1, and dgat1a in tango2 mutants ( Figure S3B).
Moreover, caffeine or mechanical loading further led to a decrease in all the enzymes in the glycerolipid pathway, including gpat3, that exhibited normal levels without any extrinsic trigger. Therefore, the overall abundance of major membrane and cellular lipids synthesized through ER/SR is significantly decreased in tango2 mutant zebrafish.

Discussion
Rhabdomyolysis Although tango2 mutants exhibit normal swimming behavior, skeletal muscle showed a disorganized myotome and a small number of abnormal mitochondria and SR. This suggests that Tango2 deficiency results in intrinsic defects in skeletal muscle structure and function; still, the basal threshold function of the skeletal muscle is sustained.
However, under certain stress conditions, these defects may prevent skeletal muscle from functioning beyond a basal threshold or may result in muscle breakdown and other abnormalities. This is evident from caffeine exposure or mechanical loading of skeletal muscle that reduced motor function and increased myofiber damage in tango2 mutants compared to controls. Mechanical loading resulted in extensive sarcomere disorganization with damaged and broken myofibers in the severely affected tango2 mutants. Prolonged mechanical loading in normal skeletal muscle can result in overstretched sarcomeres, myofibril misalignment, and myofiber atrophy. Upon rest, normal skeletal muscle undergoes rapid restructuring and recovery (Krippendorf andRiley, 1994, Newham et al., 1983). Unlike control larvae, tango2 mutants failed to restore normal skeletal muscle structure. This is similar to the exertional rhabdomyolysis commonly observed in Rhabdomyolysis patients (Carneiro et al., 2021 (Aldrich et al., 2021). Finally, this results in the activation of calcium-dependent proteases and phospholipases that contributes to the damage to myofibrillar and cytoskeletal proteins (Scalco et al., 2016).
A similar mechanism may be contributing to skeletal muscle damage in Tango2 deficiency, as shown by the increased sensitivity of tango2 mutants to caffeine that binds to Ryr1 channels in SR and induces the release of Ca 2+ in the cytosol (Chirasani et al., 2021).
To identify the basal intrinsic defects in Tango2 deficiency, we performed lipidomics in control and tango2 mutants as TANGO2 patients-derived cell lines show abnormal accumulation of fatty acids or acylcarnitines (Schymick et al., 2022, Bérat et al., 2021. However, these findings are quite divergent and failed to provide a clear outcome due to wide clinical heterogeneity in patients' samples. Our lipidomic analysis identified a significant reduction in phospholipids and triglycerides in Tango2 deficiency. These phospholipids contained unsaturated and mono-or poly-saturated fatty acid chains with decreased large chain fatty acids (16C-38C). Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids (50% of total lipids), and a decrease in these lipid species may increase the susceptibility to membrane damage by regulating membrane stability and fluidity. Previous studies have shown that a reduction in phospholipids results in skeletal muscle myopathy (Ferrara et al., 2021), and therefore decreased amounts of phospholipids in Tango2 deficiency could underlie the muscle weakness seen in TANGO2 patients. Phospholipid synthesis occurs predominantly through the glycerol-3-phosphate pathway ( Figure 6C). LPIN1 catalyzes an essential step of this process, and mutations in LPIN1 are the most common cause of severe recurrent rhabdomyolysis through loss of cell membrane integrity and myofiber dysfunction. (Zeharia et al., 2008 (Wu et al., 2015). Reduced levels of triglycerides in tango2 fish further point to defects in glycerolipid homeostasis in Tango2 deficiency. Some TANGO2 patients also exhibit acylcarnitine accumulation, suggesting that defects in the glycerol-3-phosphate pathway in Tango2 deficiency may prevent utilization of acyl-CoA, thus leading to acylcarnitine accumulation ( Figure 6C). Acyl carnitine is normally -oxidation pathway. As mitochondrial defects are also observed in Tango2 deficiency (Figures 2 & 3), decreased metabolism of acylcarnitines may lead to its accumulation which is toxic for several organs, including the skeletal muscle, heart, and liver. We did not observe any significant changes in the acylcarnitines in Tango deficiency. As lipidomics analysis in tango2 mutants was performed in the basal state, the accumulation of acylcarnitines seen in some TANGO2 patients could be triggered by metabolic or other stress states.
While no functional defects were observed at the basal state during early larval stages in tango2 mutants, a variability in survival rate was observed during larval and juvenile stages. During the first few days of development, zebrafish embryos and larvae survive on nutrients provided by the egg yolk. However, as these animals transition to external feeding at 5-6 days post fertilization, different amounts of nutrients and swimming (exercise) may elicit variable phenotypes in affected mutants. Similarly, human TANGO2 patients have variable rhabdomyolysis onset and may be caused by differences in behavior or metabolic processes. This is further evident from recent studies that showed nutrient stress controls lipid homeostasis in TANGO2 disease pathogenesis through the regulation of acyl-CoA by phosphatidic acid (Lujan et al., 2023). In another recent study, treatment with Vitamin B5, a Coenzyme A precursor, rescues seizures in a Drosophila model of tango2 deficiency (Asadi el al., 2023). This suggests that Tango2 deficiency results in cellular and membrane lipids defects in the basal stage that are exacerbated by extrinsic signals such as nutrient stress, caffeine, and exercise and are potential risk factors for developing metabolic crisis and rhabdomyolysis in TANGO2 patients. Future studies on how Tango2 regulates these processes will further improve our understanding of TANGO2-related disorders.

Zebrafish lines
Fish were bred and maintained using standard methods as described (Westerfield, 2000). All procedures were approved by the Brigham and Women's Hospital Animal Care and Use Committee. tango2 bwg210 and tango2 bwg211 zebrafish lines were created in our laboratory by the CRISPR-Cas9 approach. Zebrafish embryonic (0-2 days post fertilization) and larval stages (3-45 dpf), juvenile stage (45 dpf-3months), and adults (3 months) have been defined as described previously (Kimmel et al., 1995). Zebrafish clutches exhibiting >10% lethality (0-1 days post fertilization) were excluded from the study. All studies presented in this work were performed on tango2 bwg211 mutants obtained from heterozygous parents unless specified.

Genotyping Assays for tango2 lines.
DNA was extracted from zebrafish larvae or fin clips of adult zebrafish, genotyped by PCR, and analyzed by a 2% agarose gel (Bennett et al., 2018). PCR Primer sequences used for genotyping are 5'TGGGAATTAGCAAACGAGGA3' and 5'ATGGCTGAAAGAGCTGTGCT3'.

Rt-PCR and cDNA sequencing in controls and mutant tango2
cDNA was analyzed by gel and Sanger sequencing using heterozygous siblings as controls to detect the presence of maternal mRNA in wild-type and mutant siblings. qRT-PCR was performed using Sybr green assay as described previously (Bennett et al., 2018).

Myofiber isolation and immunofluorescence
Myofibers were isolated from control or tango2 larval zebrafish (45 dpf) as described previously with minor modifications (Ganassi et al., 2021). Skinned zebrafish muscle (Thermofisher Scientific). Imaging was performed using a Nikon Ti2 spinning disk confocal microscope and colocalization analysis was performed on Z-stack projections.

Caffeine and Dantrolene treatment
Zebrafish (6dpf) obtained from heterozygous matings were placed in individual wells of a 48-well dish, and swimming behavior was analyzed at the basal level. Caffeine and dantrolene treatments were performed as previously described with some modification (Endo et al., 2022). E3 water was replaced with 0.5uM caffeine-containing E3 water, and fish were incubated for 1 hour. Subsequently, caffeine was replaced with normal E3 water. Swimming behavior was analyzed again after an hour of recovery and after 24 hours of recovery by the automated tracking system. For dantrolene (Millipore Sigma D9175) treatment, zebrafish were incubated with 5uM dantrolene for 2 hours, followed by 1 hour incubation with caffeine and dantrolene. Swimming behavior was analyzed before the treatment, after an hour of recovery, and after 24 hours of recovery by the automated tracking system.

Methylcellulose Assay and Whole-mount phalloidin staining
Zebrafish larvae (7 dpf) obtained from tango2 heterozygous matings were individually placed in 48 well dishes in E3 water, and swimming behavior was quantified by using the Zantiks MWP automated tracking system (Zantiks Ltd., Cambridge, UK). Subsequently, E3 water was replaced with 1% methylcellulosecontaining E3 water with the E3 water, and swimming behavior was quantified again (Zantiks Ltd., Cambridge, UK). Larval heads were collected for genotyping, and bodies were fixed Disease Models & Mechanisms • DMM • Accepted manuscript in 4% paraformaldehyde. Whole-mount phalloidin staining was performed as previously described (Casey et al., 2023).

Phosphatidylcholine Quantification assay
Phosphatidylcholine quantification was performed on 8 and 30 dpf control and tango2 mutants using the Phosphatidylcholine assay kit (#MAK040, Millipore Sigma) according to the manufacturer's instructions. Briefly, pooled 30 control or mutant larval fish (7or individual fish (30dpf) were homogenized in Phosphatidylcholine (PC) assay buffer, and the supernatant was collected after centrifugation. PC hydrolysis enzyme, PC development mix, and fluorescent peroxidase substrate were added to the tissue extracts in PC buffer and incubated for 30 minutes at room temperature. The fluorescence intensity was measured (λ ex = 535/λ em = 587 nm). Data was normalized with the total body weight of each sample.

Lipidomic Profiling
Control and tango2 mutant zebrafish (4 weeks, n=5 each) were homogenized with 1 ml of MBTE. 300 uL of methanol with internal standard was added, and samples were mixed for 10 minutes. 200 uL of water was added to facilitate phase separation. The extracts were centrifuged at 20,000 rcf for 10 minutes. Avanti using internal standards and the body weight of individual samples. Samples were normalized, and biological replicates were averaged. P-value and fold change was calculated as instructed, as previously described by (Aguilan et al., 2020). P-value was set to 0.05.

Zebrafish locomotion assay
Zebrafish swimming behavior was quantified using the Zantiks MWP automated tracking system (Zantiks Ltd., Cambridge, UK). Larval zebrafish (5-7 dpf) were placed individually by randomization into each well of a 48-well plate, and their swimming behavior was recorded for 50 minutes (10 minutes light, 10 minutes dark, 10 minutes light, 10 minutes dark, 10 minutes light, end). Four independent blind trials were performed, and the total distance and cumulative duration of the movement were recorded.

Quantification and statistical analysis
All samples were blinded till final analyses, and statistical analyses were performed using GraphPad Prism9.

Materials Availability
Newly created zebrafish lines and plasmids generated in this work are available on request.

Data Availability
Lipidomics data has been deposited to the Metabolomics workbench and is available at   Quantification of transcripts of ER-localized glycerol-3-Phosphate pathway enzymes in the basal state, on caffeine exposure (C) or exercise by mechanical loading (E) at 8 dpf.