Leptin Pufferfish
Description
Introduction to Leptin Pufferfish
Leptin pufferfish, or pufferfish leptin (pLep), is a hormone found in the pufferfish (Takifugu rubripes), which plays a crucial role in regulating energy homeostasis, appetite, and metabolism. Despite its low amino acid sequence identity with mammalian leptins, pLep exhibits a conserved tertiary structure, suggesting functional similarities across vertebrates .
3.1. Metabolic Effects
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Lipid Metabolism: Studies in fish suggest that leptin can promote lipolysis and decrease lipogenesis. For example, in goldfish, human leptin injections reduced hepatic lipid content .
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Glucose Metabolism: While specific data on pufferfish leptin's role in glucose metabolism is limited, leptin in fish generally influences glucose utilization and lipogenesis .
3.2. Reproductive and Growth Effects
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Reproductive Axis: Leptin in fish, including pufferfish, may influence the reproductive axis by stimulating luteinizing hormone (LH) release, similar to its role in mammals .
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Growth Regulation: Leptin affects growth by modulating energy balance and nutrient partitioning, though specific studies on pufferfish are scarce .
Comparative Analysis with Mammalian Leptin
| Characteristics | Mammalian Leptin | Pufferfish Leptin (pLep) |
|---|---|---|
| Primary Structure | High sequence identity among mammals | Low amino acid identity with mammalian leptins (~13.2%) |
| Tertiary Structure | Four α-helices | Conserved tertiary structure with four α-helices |
| Production Site | Adipose tissue | Liver |
| Metabolic Role | Regulates appetite, energy balance, and lipid metabolism | Similar roles in appetite regulation and energy metabolism |
| Reproductive Influence | Stimulates LH release | Potential influence on reproductive axis |
Product Specs
ALPGALDAMDVEKMKSKVTWKAQGLVARIDKHFPDRGLRFDTDKVE
GSTSVVASLESYNNLISDRFGGVSQIKTEISSLAGYLNHWREGNCQE
QQPKVWPRRNIFNHTVSLEALMRVREFLKLLQKNVDLLERC
Q&A
When was leptin first identified in pufferfish?
Leptin was first identified in pufferfish (Takifugu rubripes) by Kurokawa et al. in 2005, representing one of the earliest leptin gene identifications in teleost fish . This discovery came over a decade after the initial identification of leptin in mammals by Zhang et al. in 1994, who characterized it as a 16 kDa protein originally identified in mammalian adipose tissue .
How does pufferfish leptin compare structurally to mammalian leptin?
Pufferfish leptin, like other non-mammalian leptins, shares relatively low primary sequence identity (10-30%) with mammalian leptin . Despite this low sequence homology, tertiary structure and some functional aspects of leptin appear to be conserved across vertebrates. This structural conservation explains how leptin maintains its signaling capability despite sequence divergence. The tertiary structure conservation is particularly evident in the binding interface between leptin and its receptor .
Which tissues express leptin in pufferfish?
In pufferfish and other teleosts, the liver appears to be the primary organ for leptin expression, which represents a significant difference from mammals where adipose tissue is the main production site . This distinct expression pattern suggests divergent evolutionary paths for leptin's regulatory mechanisms across vertebrate lineages.
Why does tissue expression differ between fish and mammals?
The differential tissue expression likely reflects both the evolutionary distance between teleost fish and mammals and the adaptation to different physiological needs. In mammals, leptin primarily functions as an adiposity signal, while in fish, its roles may be more diverse, including involvement in metabolism, reproduction, and possibly osmoregulation. The predominant expression in fish liver suggests leptin may function more as a metabolic regulator than purely as an adiposity signal in these species .
How many forms of leptin exist in pufferfish and other fish?
Gene duplication events in the teleost lineage have led to the expression of multiple leptin proteins in fish species. Many teleosts, including zebrafish and Japanese rice fish (medaka), possess at least two distinct leptin genes (leptin-a and leptin-b) . This contrasts with the single leptin gene found in mammals and most terrestrial vertebrates. In some fish species, such as salmon, additional genome duplication events may have resulted in even more leptin gene copies .
What is the phylogenetic relationship between pufferfish leptin and other vertebrate leptins?
Phylogenetic analyses indicate that fish leptins diverged from terrestrial vertebrate leptins after the split between aquatic and land animals. An ancient duplication of the leptin gene (resulting in leptin-a and leptin-b) occurred in the teleost lineage, followed by at least one additional genome duplication event in some fish lineages . This evolutionary history explains the greater diversity of leptin forms in fish compared to mammals and other terrestrial vertebrates.
Does leptin regulate appetite in pufferfish similar to mammals?
The role of leptin in appetite regulation in pufferfish and other teleosts remains somewhat controversial. While leptin is established as a potent anorexigenic signal in mammals, studies in various fish species have shown contradictory results, likely confounded by factors such as leptin source (native or heterologous), method of delivery (intraperitoneal or intracerebroventricular), dosage, and experimental conditions . Some studies in other fish species suggest leptin may play a role in appetite regulation by influencing the expression of orexigenic and anorexigenic genes in the brain, but the mechanisms may differ from those in mammals .
What other physiological processes might pufferfish leptin influence?
Beyond potential appetite regulation, leptin in fish may play pleiotropic roles in various physiological processes including:
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Osmotic adaptation and osmoregulation
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Glucose homeostasis and metabolism
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Stress regulation
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Reproductive function
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Metabolic rate regulation
Evidence from zebrafish studies demonstrates that leptin signaling influences metabolic rate in fish embryos, suggesting conservation of this function across vertebrates .
Do different fish leptin forms have distinct binding properties?
Computational analyses predict different binding energies between leptin forms and their receptors in fish species. In zebrafish and Japanese rice fish, leptin A is predicted to have a higher binding energy to the leptin receptor than leptin B . These differences in binding energies suggest either:
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Divergent functions for the different leptin forms
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Different binding conformations
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Potential interactions with other protein partners beyond the canonical leptin receptor
What experimental techniques are most effective for studying leptin function in pufferfish?
Several experimental approaches have proven valuable for investigating leptin function in fish:
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Gene knockdown/knockout studies: Antisense morpholino oligonucleotide technology has been used to knockdown leptin gene expression in developing zebrafish embryos to assess its effects on physiological processes such as metabolic rate . CRISPR-Cas9 gene editing can be employed for creating stable leptin knockout lines.
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Recombinant protein administration: Administration of recombinant leptin (either native fish leptin or heterologous mammalian leptin) via intraperitoneal (IP) or intracerebroventricular (ICV) injection allows for assessment of acute leptin effects.
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Gene expression analysis: Quantitative PCR and RNA sequencing help identify downstream targets of leptin signaling and tissue-specific expression patterns.
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Structural modeling and protein interaction studies: Computational approaches to model leptin-receptor interactions provide insights into binding mechanisms and evolutionary conservation .
How can researchers address the challenges of working with multiple leptin forms?
When studying leptin in pufferfish and other teleosts with multiple leptin forms, researchers should:
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Design paralog-specific primers and antibodies that can distinguish between leptin A and leptin B.
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Consider potential compensatory mechanisms when one paralog is knocked down or knocked out.
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Perform comprehensive expression profiling to determine tissue-specific and developmental stage-specific expression patterns of each paralog.
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Conduct comparative functional assays to determine potentially distinct roles of each leptin form.
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Use recombinant proteins of each specific form rather than heterologous (e.g., mammalian) leptin for functional studies whenever possible .
How consistent is leptin function across teleost species?
While certain aspects of leptin function appear conserved across teleosts, significant species-specific variations exist. Studies in different fish species have revealed sometimes contradictory results regarding leptin's effects on appetite regulation. For example, after leptin administration, expression levels of proopiomelanocortin (pomc) increased in goldfish and rainbow trout but decreased in mandarin fish . These variations highlight the importance of species-specific studies rather than generalizing findings across all teleosts.
What might explain the temperature-dependent functions of fish leptin?
Fish leptin proteins show greater variation in hydrophobic amino acids compared to mammalian leptins, which may contribute to temperature-dependent functions . Since fish live in variable temperature environments unlike endothermic mammals, their leptin-receptor interactions may have evolved to function optimally across a range of temperatures. For example, zebrafish show a greater number of hydrophobic amino acids in the leptin-receptor interaction interface relative to humans, suggesting more temperature-controlled interactions .
What are the major technical challenges in pufferfish leptin research?
Several challenges complicate research on pufferfish leptin:
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Limited availability of species-specific antibodies and assays
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Challenges in expressing and purifying correctly folded recombinant fish leptin proteins
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Difficulty in maintaining consistent experimental conditions, particularly temperature, which may affect leptin function
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Potential functional redundancy between multiple leptin forms
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Limited tools for genetic manipulation in pufferfish compared to zebrafish
What are promising future research directions for pufferfish leptin?
Future research on pufferfish leptin should focus on:
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Comprehensive characterization of differential functions between leptin A and leptin B
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Identification of novel leptin-responsive pathways unique to fish
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Investigation of temperature-dependent leptin signaling mechanisms
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Exploration of potential roles in processes like osmoregulation and reproduction
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Comparative studies across fish species living in different environments to understand adaptive significance of leptin variation
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Development of pufferfish-specific genetic tools to facilitate functional studies
Product Science Overview
Leptin is a peptide hormone primarily produced by adipose tissue in mammals. It plays a crucial role in regulating energy balance by inhibiting hunger, which in turn diminishes fat storage in adipocytes. The hormone communicates the status of energy reserves to the brain, particularly the hypothalamus, thereby influencing appetite and metabolic rate .
The leptin gene was first identified in mammals in 1994 by Jeffrey Friedman and his team. Since then, leptin genes have been discovered across various vertebrate species, including fish, amphibians, reptiles, and birds . The leptin protein structure has been conserved throughout evolution, although there are significant differences in the primary amino acid sequences among different species .
The pufferfish (Takifugu rubripes) leptin gene was identified and cloned to study its structure and function. The recombinant leptin from pufferfish is produced in Escherichia coli as a single, non-glycosylated polypeptide chain with a molecular mass of approximately 16 kDa . This recombinant leptin is bioactive and has been used in various studies to understand leptin’s role in fish physiology.
The preparation of recombinant leptin involves cloning the leptin gene from pufferfish into a suitable expression vector, which is then introduced into Escherichia coli. The bacteria are cultured, and the leptin protein is expressed, harvested, and purified. The resulting product is a sterile, filtered, white lyophilized (freeze-dried) powder .
Recombinant leptin from pufferfish has been instrumental in studying the hormone’s role in non-mammalian species. It has provided insights into the evolutionary conservation and divergence of leptin’s function across different vertebrate classes. Additionally, understanding leptin’s role in fish can have applications in aquaculture, where managing energy reserves is crucial for the growth and health of commercially important species .
