In this study, the jaw fat transcriptome data was obtained from OJFs and IJFs of three male Risso’s dolphins for minimum statistical limitations and analyzed by high throughput NGS technology to understand the genetic lineages of metabolism of these specialized acoustic fats. We believe that identifying the fat metabolism of these important animals may contribute industrial applications for biosynthesis and protecting aquatic wildlife.
We first identified the DEGs in both jaw fat tissues based on total transcriptome data and analyzed if for functional annotation. Illumina produce short readings with an average length of 500 bp in large numbers of copies which are annotated to the reference genome to identify genes. However, RNA-seq technology is still being developed in order to perform long-read sequencing 17. Three biological replicates have used to optimize transcriptome data, and edgeR was used as a differential expression tool for false positive performances, as recommended 18,19. High quality and accurate sequenced data were obtained from this study for further analysis. Cuffdiff is also a supportive tool for assembling readings from two or more biological conditions and identify DEGs for transcriptional analyses 20.
The two jaw fats of toothed whales, IJF and OJF, showed several differences based on previous studies 8,21–23. The OJF is located around the lower jaw of toothed whales and directly connected with other tissues; however, IJF is present inside the lower jaw hole. Therefore, the mechanism of generation for these two fats is certainly a concept worthy of study. According to the previous findings, there is a phylogenetic influence for the development of these acoustic fats and endogenous synthesis 21,24. Additional studies have emphasized that sound transmission through the fat bodies is possible and may alter the wavelength physically and change depending on the species, thereby reducing competition over predation. However, detailed studies are still needed to explain this unknown puzzle. We also recorded differences in upregulated DEGs by bioinformatics tools in both of the fat tissue types; to our knowledge, this observation is the first of its kind. These findings indicate that those DEGs are related to lipid metabolism activities. A higher number of DEGs were observed in the outer fat compared to the inner fat.
Interestingly, fatty acid binding protein 1 (FABP1), apolipoprotein H (APOH), fructose-bisphosphate aldolase B (ALDOB), zinc finger protein 37A (ZN37A), ALBU, solute carrier families 1 and 2 (SLC1A2 & SLC2A2) and hepatocyte nuclear factor 4, alpha (HNF4A) were identified in the OJF, which may directly engage with lipid metabolism. A study has revealed the FABP4 is associated with fat deposition in bovine populations 25. The FABP1 is a highly-expressed DEG in the OJF and downregulated in IJF in the current study. The APOH protein is involved with triggering of lipoprotein lipase in lipid metabolism 26,27. The ALDOB gene is an important gene in mammals’ metabolisms and homeostasis. This gene contributes to fructose metabolism and, therefore, enhances glycogen and lipid synthesis 28. ZN37A is one of zinc finger proteins (ZFPs), a direct study on this lipid’s metabolism has not been found, however ZFPs are involved with lipid bindings and lipid and glucose metabolism 29,30. ALBU is also identified as a biomarker for lipid-related diseases in human-like obesity 27,31. SLC1A2 and SLC2A2 are solute carrier associated proteins which are involved in lipid biosynthesis, fatty acid metabolic process and fatty acid transport 32–35. HNF4A is a transcription factor that binds with fatty acids 36, regulates the expression of apolipoproteins 37 and is involved with lipid metabolism 38. On the other hand, the IJF contains DEGs of amyloid beta precursor protein (APP), Thrombospondin-4 (TSP4), mRNA decay activator protein (ZFP36L1) and ATP-dependent RNA helicase A (DHX9). These genes share some relationships with lipid metabolism such as the APP and TSP4 genes which are involved with bone marrow adiposity 39–41; DHX9 shows an oxidative stress responsiveness 42. Another study has identified a complex bidirectional link between lipids and APP related to lipid alteration, APP processing and regulation of lipid metabolism pathways in brains 43.
We also checked the expression levels of common genes of TAG/WAX metabolism KEGG pathways (Table 4) to acquire a comprehensive biological insight from the enrichment of expressed genes. Interestingly, FAS, malonyl CoA-acyl carrier protein transacylase (FabD), carbonyl reductase family member 4 (CBR4), hydroxysteroid 17-Beta Dehydrogenase 8 (HSD17B8), hydroxyacyl-thioester dehydratase type 2 (HTD2), malonyl CoA-acyl carrier protein transacylase (MCAT) of the fatty acid synthesis, branched-chain amino acid aminotransferase/transaminase (BCAT2), acetolactate synthase I/II/III large subunit (ilvl) of the valine, leucine and isoleucine biosynthesis pathway, BCAT2 and wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase (WS/DGAT) of the valine, leucine and isoleucine degradation pathways were more expressed in the IJF than the OJF. However, fatty acid binding protein (FABP5) and acyl carrier protein (ACP), of fatty acid synthesis pathway and aldehyde dehydrogenase (ALDH2) of the valine, leucine and isoleucine degradation pathways, were increasingly expressed in the OJF compared with the IJF.
Based on this diversity of DEGs in both jaw fat tissues, we suggest that there is a clear possibility of endogenous synthesis of these specialized fats in particular locations of the lower jaw area of toothed whales. Therefore, we concur with the previous suggestion of previous experts (Koopman, 2018): The evolution of unique de novo lipid biosynthesis pathways to are used produce wax esters and short branched-chain pathways in the suborder Odontoceti (toothed whales).
In this study, we used Enrichr, the online tool of the Ma’ayan lab, mainly for the functional enrichment analysis using the DEG sets in the two tissue types. The specialties of this tool are a large collection of diverse gene libraries, improved programming interface and a variety of visualization choices, fuzzy enrichments, easy to use, open source and ranking enriched terms with p value 44,45. The functional enrichment analysis contains two categories such as a GO analysis and a pathway analysis, both of which were conducted using Enrichr. GOs were comprised with several groups, however we considered four main categories such as biological, cellular, molecular and Jensen. Pathway enrichments were also categorized into several groups. In this study, we used KEGG 2019 human, MGI-Mammalian, BioPlanet 2019 and WikiPathways 2019 to identify reliable pathways. The selection of an updated database for functional enrichments is highly advised 46; therefore, we believed a collection of information from several database may provide accurate results. We mainly focused 23 terms related to the lipid metabolisms for further discussions to prove our hypothesis.
In the OJF (Table 1), the main lipid related identified biological processes were phospholipid homeostasis (GO:0055091), positive regulation of lipoprotein lipase activity (GO:0051006), positive regulation of triglyceride lipase activity (GO:0061365), regulation of cholesterol homeostasis (GO:2000188), regulation of lipoprotein lipase activity (GO:0051004), acylglycerol homeostasis (GO:0055090), glycolytic process (GO:0006096) and triglyceride homeostasis (GO:0070328). Phospholipid homeostasis is involved with regulation of phospholipids in organisms and specially in mammals, it can increase curvature elastic stress which may contribute to de novo lipid synthesis 47. The positive regulation of lipoprotein lipase activity is also relatively observed in this tissue. This supports the hydrolysis of triacylglycerol and the uptake of free fatty acids from the plasma 48. The activities to increase triglyceride lipase in OJF has also enriched. Angiopoietin-like proteins, circulating triglyceride (TG) levels 49 and peroxisome proliferator-activated receptors (PPAR-gama) can regulate adipose triglyceride lipase in adipocytes 50. Cholesterol homeostasis and fatty acid metabolism can be regulated by liver X receptors (LXR), and PPAR-gama can also induce expression of ATP binding cassette transporters (ABCA) which is essential for lipid raft maintenance and apolipoproteins by upregulating LXR 51,52. Multifunctional O-acyltransferase can catalyze the acylglycerol homeostasis, waxes and retinyl esters which are important in lipid metabolisms 53. The overexpression of acylglycerol, which catalyze triacylglycerol formation 54. Glycolytic process triggers glycolytic flux which regulates the fatty acyl-CoAs 55. Triglyceride homeostasis is the most important enriched ontology in this tissue, which is directly relate with the lipid metabolism. Triglycerides and cholesterol esters can store in lipid droplets and can contribute to de novo lipid synthesis 56. The triglyceride-rich lipoprotein particle is also enriched in the Jensen compartments. The lipid metabolism-related GO cellular component, a very-low-density lipoprotein (VLDL) particle (GO:0034361), was enriched in the OJF. This is also highlighted in the Jensen compartments. VLDLs are composed of triacylglycerols and cholesterols which are produced from liver and travel via plasma and can hydrolyze to provide fatty acids in a particular organ 57,58. In relation to the VLDLs, high-density lipoprotein particles are also enriched according to the Jensen compartments. The lipase activator activity (GO:0060229) is the only enriched molecular GO term in the OJF, whereas lipoprotein lipase is highly important for lipid metabolism in animals 59.
Pentose phosphate pathway, hepatocyte nuclear factor 3-beta (HNF3B) pathway, glycolysis and gluconeogenesis, facilitative sodium-independent glucose transporters, developmental biology (white adipocyte differentiation), glutathione conjugation, glycolysis, gluconeogenesis, glycolysis and gluconeogenesis WP534 and Cori Cycle WP1946 were the enriched terms in OJF according to KEGG, BioPlanet and WikiPathways. A study has revealed that an oxidative pentose phosphate pathway can doubly enhance fatty acid biosynthesis 60, and this pathway can provide NADH to accelerate the production of acetyl-CoA, a precursor of fatty acids 61. Acceleration of an HNF3B pathway can activate genes related to glucose homeostasis, therefore indirectly influencing lipid metabolism 62. Glycolysis and gluconeogenesis are other enriched pathways that are involved in synthesizing lipids; fatty acid metabolism may contribute to de novo fatty acid synthesis 63. Facilitative sodium-independent glucose transporters are an active pathway in OJF, they are responsible for the absorption and distribution of glucose and other sugars in mammals 64. Moreover, BioPlanet database has identified the white adipocyte differentiation in the nuclear receptor PPAR-gama may play an important role in this activity (Niemala et al., 2008; Mueller, 2014). The glutathione conjugation pathways also has a strong affinity for glutathione-derivatized fatty acids 67. The Cori cycle is also involved within the gluconeogenesis, therefore it may be responsible for the metabolism in the liver and related to lipolysis in adipose tissues 68.
In IJF, lipid metabolism-related GO enrichment biological terms are shown in the Table 2. The positive regulation of lipid-binding terms comes under the positive regulation of binding; therefore, it is associated with lipid metabolism. ALBP (adipocyte lipid binding protein) includes an intracellular lipid binding family 69 which is involved in delivering fatty acids to appropriate sites for energy and regulatory processes 70. The APP is an important gene found in IJF that is responsible for many functional enrichment terms, especially positive regulation of amyloid-beta formation. Amyloid-beta is directly involved with lipid homeostasis, and many lipids are involved with amyloid-beta lipid regulatory system bidirectionally 71. Serum amyloid A3 (SAA3) is also an example that was recently found which contributes to lipid deposition in mammals 72. Also, according to another recent finding, plasma lipids and plasma amyloid-beta has a functional relationship 73. Activation of pyroptosis is supported with monounsaturated oleic acid and lipotoxicity 74 and upregulated adipogenesis and misbalanced fatty acid metabolism 75. Transport along microtubule is also enriched in IJF. In many cells, lipid droplets get active movement through microtubules for various purposes like biogenesis 76. Microtubules play an important role in the cell to bidirectional transport of cellular cargos 77. Positive regulation of lymphocyte migration is another enriched term, for example, immune cells interact with lipids and control plasticity and T lymphocytes 78. Cytotoxic T lymphocytes has relation with maintaining high level of proteins and lipid synthesis 79. Positive regulation of T cell migration is also effective on lipid metabolism such as glycolysis, fatty acid and glycosphingolipid metabolism 80. Positive regulation of nucleocytoplasmic transport in mesenchymal stem cells may involve with adipocyte generation 81. Glial cell development has enriched in IJF, these cells can accumulate of lipid droplets 82. Another study has revealed that lipid-mediated communication between glial cells and neurons, however lipid metabolisms in glial cells is still not clear 83,84. The IJF is known as the receiver of echolocated sound waves, therefore cell-cell communication might be a good evidence for further investigations. Moreover, lipoprotein-dependent lipid accumulation may have relation with the origin of glial cell formation in vertebrates 85. Lipids in cells can regulate cation channel activity in different ways such as fatty acids change membrane mechanics, regulate, interact and modify ion channel functions 86. Protein localization to organelle was a GO-enriched term in IJF. Phospholipids are important transporters of lipids from endoplasmic reticulum to other organelles 87. On the other hand, transport can vary significantly with the type of the cell and some organelles have lipids to go along with unique proteomes 88. Peroxisome is a cellular enriched term in IJF which is direct nexus for lipid synthesis, fatty acid oxidation, production of ether lipids and cellular signaling 89,90. Furthermore, lipid droplets and peroxisomes maintain lipid homeostasis in cells 91,92. Microbodies are associated with lipid globules and make microbody-lipid globule complex 93. Lipid metabolism is integrated with Golgi function, membrane raft and control cellular trafficking 94,95. Axon is another important cellular enriched term in the IJF. Axon can be involved with lipid transportation intracellularly or intercellularly and axon to myelin, however its pathways are still unknown 96. Lipids are also involved with the regeneration and elongation of axons 97–99. Integrin binding is the only enriched molecular term of the IJF. The integrin function can be regulated by lipid rafts 100. Nuclear envelope lumen is the region in between two lipid bilayers, which maintains traffic between the nucleus and cytoplasm 101, and it is involved with lipid metabolisms 102. A study has identified that inner nuclear membrane helps to generate nuclear lipid droplets 103. Cilia are also involve with protein localization in the cell, and lipid modifications properly direct these proteins 104.
ECM-receptor interaction, peroxisome, serotonergic synapse, copper homeostasis and advanced glycosylation end-product receptor signaling are the main enriched pathways in the IJFs. The extracellular matrix (ECM) receptor interaction is involved with lipid metabolism, adipogenesis and maintain tissue architecture 105. Integrins play an important role between cells and ECM while significant enrichment has observed in subcutaneous and intramuscular fats in cattle 106,107. This pathway also recorded as enriched in intramuscular fat metabolism between breast and thigh tissues of chickens 108, metabolism of fats in tails of sheep 109. Peroxisomes are also enriched KEGG pathway in IJFs and it is also enriched as a cellular GO term as we described in the earlier paragraph. However, we can further confirm that as a key metabolic organelle, peroxisomes are involved with lipid metabolism for beta-oxidation of fatty acids and synthesis of myelin sheath lipids 110. Serotonergic synapse is a enriched KEGG pathway in this tissue that express the function of lipids as a neurotransmitter 111,112 and controlling metabolic homeostasis 113. Copper homeostasis WP3286 is a enriched term from WikiPathways that refers to the IJF, clearly saying that importance of copper for the lipid synthesis and insufficient copper may cause for lipid related diseases 114. Copper also play a role as a modifier in lipid metabolism and adipocytes require copper to balance the metabolic fuels and de novo lipogenesis 115. Advanced glycosylation end-product receptor signaling pathways have some correlations with inflammation 116, cellular signaling 117 and adipogenesis 118.
Comparing all the enriched DEGs and functional terms between inner and OJFs, we predict that two different lipid synthesis and metabolism patterns have developed in inner and OJFs in toothed whales. According to the expression levels of common genes in KEGG TAG/WAX synthesis pathways (Table 4), we can clearly identify that valine, leucine and isoleucine biosynthesis and degradation can occur in both fat tissues. The normal fatty acid synthesis also can happen in the inner fat with presence of all genes in the fatty acid synthesis KEGG pathway, but it may not manifest in the OJF. The presence of wax ester in mammalian adipose tissues is not common and may be specified only to toothed whales. The intake of wax esters from diets is impossible, therefore it was strongly suggested that wax in toothed whales should be de novo synthesis endogenously 6. By accepting this suggestion, our study presents a wax ester synthesis gene, WS/DGAT, in both IJF and OJF tissues (Figure 5). However, DEGs and functional enriched terms in the OJF represented a possibility of biosynthesis of fatty acids, maybe in a unique pathway. More importantly, we came to a prediction based on the presence of the APOH gene in the OJF and the APP gene in the IJF. The metabolism of lipids in the outer jaw can be mainly mediated with the APOH protein as a transporter of lipids, and this is supported by other studies 119,120. Meanwhile, APP might be involved in exchanging lipids from lower jawbone marrow fat and de novo synthesis within the IJF tissue. Specifically, enriched terms in the IJF support the creation of a new hypothesis: These fats may be able to transfer the receiving echolocation signals to the brain via neurons with the activation of neurotransmitters (Figure 6). Hence, APP might be an echolocation gene in toothed whales, but this still needs more research to be confirmed. The neural physiology of specific circuits used for echolocation in dolphins has evidenced in some studies 121. Similar symptom of hemorrhaging has been observed in central nerve system (CNS) and acoustic jaw fat in beaked whales due to sonar 122, therefore these could be the evidences to prove the link between IJFs and brain via the CNS for echolocation in toothed whales. Supporting, a study has described that presence of adipose tissue, blood vessel and nerves in the fibrous tissue of temporomandibular joint of dolphins play a role in echolocation as a neurological sensory function (McDonald et al., 2015; Bruno Cozzi; Stefan Huggenberger; Helmut Oelschläger, 2017). The involvement of neural system for acoustic motor-sensory coupling for echolocation has been investigated to human (Thaler, Arnott and Goodale, 2011; Flanagin et al., 2017) and bats (Covey, 2005; Razak and Fuzessery, 2015). The difference of DEGs between inner and OJFs may also correlate with the physiochemical difference of branched-chain lipid concentration and distribution among the two fats 6. We believed that this is the first study of a transcriptomic analysis of mandibular/jaw/acoustic fats in Odontocetes (toothed whales). Therefore, there is still a big vacuum of knowledge to compare metabolism and biosynthesis of these specialized fats. We recommend further analyses of acoustic fat tissues of toothed whales using different omics tools in order to better understand the de novo synthesis of these unique lipids.
In summary, we performed a transcriptomic analysis on lower jaw fats of the Risso’s dolphin as a representative organism of toothed whales that possess a special aquatic adaptation, their echolocation. During this study, the lower jaw fat of toothed whales has been extensively studied as a receiver of signals of echoes from a given object. However, the metabolism of these specialized fats is still largely unknown. According to a lipidomic analysis, these fats contain a combination of TAG and wax esters. The combination and distribution of these fats are different in the inner and outer lower jaw. We studied RNAs in both fats, and their DEGs were identified. Additionally, enrichment analyses were also conducted, mainly by Enricher at p<0.05. Our results indicated that 34 DEGs in OJF and nine DEGs in IJF were enriched. APOH, HNF4A, MYF6, SLC1A2, SLC2A2 and ALDOB were identified as key genes involved with lipid metabolism of functional enrichments. The main GO and pathway enrichments mainly suggested that these genes were positively related to lipoprotein lipase activities and the activation of pentose phosphate pathway in the OJF. On the other hand, IJF contains APP, DHX9, PXMP4 and THBS4 as highly expressed genes, which are responsible for lipid metabolism. These genes are mainly involved with the positive regulation of binding and amyloid-beta formation and the activation of ECM-receptor interactions. Our study also revealed the presence of wax ester synthase in both fat tissues and the possibility of fatty acid synthesis in IJF according to KEGG pathways. Moreover, we made a new hypothesis that IJF may directly transfer the receiving signals of echoes to the brain by potential activation of neurotransmitters by the APP gene; therefore, APP might be an echolocation gene. The interactions between IJFs and OJFs needs further investigation to reveal the underlying molecular mechanisms affecting the endogenous lipid metabolisms in toothed whales. These finding could be useful for understanding the industrialized biosynthesis of these specialized lipids. Therefore, more protection methods can be implemented for these animals and other conservation efforts.