Leptin-B Tilapia
Description
Functional Roles of Leptin-B in Tilapia
While most studies focus on LepA, emerging data suggest LepB has specialized roles:
Metabolic Regulation
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Glucose Homeostasis: In Mozambique tilapia (Oreochromis mossambicus), LepB modulates glycolysis by upregulating phosphofructokinase (pfk1) and lactate dehydrogenase (ldha) genes in hepatocytes, enhancing glucose catabolism .
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Lipid Metabolism: Acute LepB administration transiently increases hepatic triglyceride levels by suppressing acetyl-CoA carboxylase (acc) expression, though effects are less pronounced than in mammals .
Appetite Regulation
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Indirect evidence from mandarin fish (Siniperca chuatsi) suggests LepB suppresses food intake by downregulating hypothalamic orexigenic genes (npy, agrp) . Similar mechanisms may exist in tilapia, but direct studies are lacking.
Comparative Insights from Related Species
Leptin-Insulin Axis
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In Nile tilapia (O. niloticus), glucagon and insulin upregulate LepA expression, but LepB’s interaction with these hormones remains uncharacterized .
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Recombinant tilapia LepA (rtLepA) suppresses insulin secretion at high glucose levels, hinting at cross-regulation that LepB might share .
Stress and Energy Expenditure
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LepB may enhance glycolysis during osmotic stress (e.g., salinity changes) to meet increased energy demands, as seen in prolactin cells of O. mossambicus .
Unresolved Questions and Future Directions
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Tissue-Specific Signaling: How does LepB interact with receptors in the brain versus peripheral tissues?
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Long-Term Metabolic Effects: Most studies use acute leptin treatments; chronic effects in obese or fasted tilapia are unknown .
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Evolutionary Drivers: Why do teleosts retain two leptin paralogs with overlapping yet distinct functions?
Product Specs
Leptin, a protein hormone primarily produced by adipose cells, plays a crucial role in energy homeostasis by regulating hunger. It exerts its effects by binding to nuclear receptors located in the hypothalamus, specifically the arcuate nucleus. Similar to insulin resistance observed in type II diabetes, obesity is characterized by reduced sensitivity to leptin, leading to an inability to sense satiety despite elevated energy stores and leptin levels. Tilapia (Oreochromis niloticus) possess full-length cDNA sequences encoding two leptin isoforms, tLepA and tLepB, as well as a leptin receptor, tLepR. The tLepR cDNA, spanning 3423 bp, encodes a protein of 1140 amino acids containing all essential functional domains conserved among vertebrate leptin receptors. The tLepA and tLepB cDNAs, measuring 486 bp and 459 bp, respectively, encode proteins of 161 and 152 amino acids. Notably, the three-dimensional structures of tLepA and tLepB exhibit significant conservation with human leptin, comprising four alpha-helices.
Recombinant Tilapia Leptin-B, expressed in E. coli, is a single, non-glycosylated polypeptide chain consisting of 152 amino acids, with a molecular weight of 15,243 Daltons. The purification process involves proprietary chromatographic techniques.
Lyophilized from a concentrated solution (1 mg/mL) containing NaHCO3 at a 1:2 salt-to-protein ratio.
For reconstitution, it is advised to dissolve the lyophilized Tilapia Leptin-B in sterile water or 0.4% NaHCO3 adjusted to a pH of 8-9, at a concentration not less than 100 µg/mL. This solution can then be further diluted in other aqueous solutions as needed.
Lyophilized Tilapia Leptin-B, while stable at room temperature for up to 3 weeks, should be stored desiccated at or below -18°C. Upon reconstitution, it is recommended to store Tilapia Leptin-B at 4°C for 2-7 days. For extended storage, freezing below -18°C is advisable. The addition of a carrier protein such as 0.1% HSA or BSA is recommended for long-term storage. It is crucial to avoid repeated freeze-thaw cycles.
The purity is determined to be greater than 95.0% using the following methods:
(a) Gel filtration analysis.
(b) SDS-PAGE analysis.
Tilapia leptins have demonstrated biological activity by promoting the proliferation of BAF/3 cells stably transfected with the long form of the human leptin receptor. However, their potency is lower compared to mammalian leptin. Additionally, Tilapia leptins exhibited biological activity by inducing STAT-LUC activation in COS7 cells transfected with the Tilapia leptin receptor, but not in cells expressing the human leptin receptor. Notably, Tilapia Leptin A displayed higher activity than Tilapia Leptin B.
Protein quantification was performed using UV spectroscopy at 280 nm. An absorbance value of 0.19 for a 1 mg/mL solution of Leptin-B Tilapia at pH 8.0 was used as the extinction coefficient. This value was determined using the DNAman computer analysis program for protein sequences.
The first six N-terminal amino acids of recombinant Tilapia leptin B are Ala-Leu-Leu-Thr-Lys-Gly.
Q&A
What is Leptin-B in tilapia and how does it differ from Leptin-A?
Leptin-B (LepB) is one of two leptin paralogues found in tilapia and other teleost fish, resulting from gene duplication. Unlike mammals that possess a single leptin gene, tilapia express both LepA and LepB with distinct functional profiles. LepB plays a predominant role in food intake regulation and glucose homeostasis, while LepA shows more limited involvement primarily in gluconeogenesis and lipid metabolism . The full-length cDNA of tilapia LepB (tLepB) is 459 bp, encoding a protein of 152 amino acids, whereas tLepA is 486 bp encoding 161 amino acids .
What is the molecular structure of Leptin-B in tilapia?
Tilapia Leptin-B is a single, non-glycosylated polypeptide chain containing 152 amino acids with a molecular mass of approximately 15,243 Daltons . Despite relatively low sequence identity with mammalian leptins, the three-dimensional structure of tLepB demonstrates strong conservation of tertiary structure with human leptin, comprising four helices . This structural conservation suggests functional conservation across vertebrate evolution despite sequence divergence.
How should recombinant Leptin-B be prepared for research applications?
Recombinant tilapia Leptin-B is typically produced in E. coli expression systems. For optimal preparation, lyophilized Leptin-B should be reconstituted in sterile water or 0.4% NaHCO₃ adjusted to pH 8-9, at a concentration not less than 100μg/ml . For short-term storage (2-7 days), reconstituted protein should be kept at 4°C, while long-term storage requires temperatures below -18°C with the addition of a carrier protein to prevent adhesion to surfaces .
How does Leptin-B regulate food intake in tilapia?
Leptin-B functions as an anorexigenic hormone in tilapia by inhibiting food intake through modulation of hypothalamic orexigenic gene expression . Acute intraperitoneal (IP) administration of recombinant LepB significantly reduces food consumption in mandarin fish, which serves as a model for studying leptin function in teleosts. This effect differs markedly from LepA administration, which shows no significant impact on food intake . The anorexigenic effect of LepB appears to be evolutionarily conserved across vertebrates, though the mechanisms may differ from mammals.
What role does Leptin-B play in glucose metabolism?
Leptin-B exhibits significant involvement in glucose homeostasis regulation in tilapia. Experimental evidence shows that LepB administration decreases hepatic glycogen levels by regulating the gene expressions of glycogen synthase and glycogen phosphorylase, with effects persisting for up to 4 days post-administration . Additionally, LepB downregulates key gluconeogenic genes including phosphofructokinase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase, suggesting a role in controlling glucose production rates .
How does fasting affect leptin expression in tilapia?
Unlike in mammals where leptin levels typically decrease during fasting, studies in tilapia demonstrate that circulating LepA and leptin receptor (lepr) gene expression actually increase after a 3-week fasting period and decline to control levels 10 days following refeeding . This pattern mirrors that observed for pituitary growth hormone (GH) regulation and contrasts with hepatic GH receptor and IGF dynamics in tilapia . This response suggests leptin in fish may serve as a signal of energy deficit rather than energy surplus as in mammals.
What approaches are used to study Leptin-B function in tilapia?
Several experimental approaches have proven effective for investigating LepB function:
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In vivo administration studies: Intraperitoneal injection of recombinant LepB (typically 400 ng/g body weight) to assess physiological responses in food intake, glucose metabolism, and gene expression .
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Ex vivo tissue explant studies: Isolated hepatocytes or Brockmann bodies (pancreatic islets) are exposed to recombinant LepB to examine direct effects on gene expression and hormone secretion .
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Molecular expression analysis: qPCR measurement of leptin-responsive genes in hypothalamus, liver, and other tissues following LepB administration .
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Metabolic parameter assessment: Measurement of blood glucose, hepatic glycogen, and triglyceride levels at various timepoints post-administration .
How can researchers verify the bioactivity of recombinant Leptin-B?
Recombinant tilapia Leptin-B bioactivity can be verified through several approaches. Cell proliferation assays using BAF/3 cells stably transfected with leptin receptors provide an in vitro assessment, though tilapia leptins typically show lower activity than mammalian leptin in these systems . In vivo verification can be performed through dose-dependent assessment of blood glucose changes following intraperitoneal injection, as observed with recombinant LepA in tilapia . Additionally, analyzing the expression of known leptin-responsive genes (orexigenic genes in hypothalamus, metabolic genes in liver) following administration can confirm bioactivity .
What are the considerations for designing loss-of-function experiments for Leptin-B research?
When designing loss-of-function experiments:
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Receptor mutant models: Loss-of-function leptin receptor zebrafish strains (lepr sa12953) have been developed and can be used as models for studying teleost leptin function .
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Paralog specificity: Due to the presence of two leptin paralogs, researchers must carefully distinguish between LepA and LepB effects through paralog-specific knockdown approaches.
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Compensatory mechanisms: Consider potential compensatory upregulation of other pathways during chronic leptin signaling deficiency.
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Physiological parameters: Monitor multiple parameters including food intake, growth, glucose homeostasis, and energy metabolism to capture the diverse functions of LepB.
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Experimental timing: Assess responses under various nutritional states (normal feeding, fasting, refeeding) to capture condition-dependent effects .
How does the leptin-insulin axis function in tilapia?
Evidence supports the existence of a leptin-insulin axis in tilapia, though with distinctive characteristics compared to mammals. In tilapia:
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Insulin increases leptin A expression 2.5-fold in vitro and 70-fold in vivo
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Glucagon increases leptin A expression in vitro and in vivo (18-fold higher than controls)
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Recombinant tilapia LepA influences insulin secretion in a glucose-dependent manner, decreasing insulin at basal glucose levels while increasing it at high glucose concentrations
This bidirectional communication suggests an evolutionarily conserved leptin-insulin axis that may be fundamental for nutrient balance regulation across vertebrates, though the relationship appears more dynamic in teleosts compared to the more tightly regulated mammalian system .
How do the functional roles of LepA and LepB differ in tilapia metabolism?
LepA and LepB exhibit distinct functional profiles in tilapia metabolism:
These functional differences suggest subfunctionalization following gene duplication, with each paralog evolving specialized roles in energy homeostasis.
What are the tissue-specific expression patterns of leptin genes in tilapia?
In Nile tilapia, tissue distribution analysis reveals distinct expression patterns:
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LepA mRNA shows highest expression in liver, with the second highest expression surprisingly in skin
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Immunohistochemical analysis confirms LepA protein expression in both liver and subcutaneous adipose tissue (SCAT)
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This pattern differs from mammals where adipose tissue is the primary leptin production site
The high expression of leptin in both liver and SCAT suggests that in tilapia, unlike mammals, multiple tissues contribute significantly to circulating leptin levels, potentially reflecting evolutionary adaptation to aquatic environments and different energy storage strategies .
How has the function of Leptin-B evolved in teleost fish compared to mammalian leptin?
Several key evolutionary divergences are evident when comparing teleost Leptin-B with mammalian leptin:
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Gene duplication: Teleosts possess two leptin paralogs (A and B) with specialized functions, while mammals have a single leptin gene .
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Response to nutritional status: Unlike mammals where leptin decreases during fasting, teleost leptin expression may increase during fasting, suggesting an inverse relationship with energy stores .
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Primary production sites: Mammalian leptin is primarily produced by adipose tissue, while teleost leptins show highest expression in liver, with significant expression also in skin and subcutaneous adipose tissue .
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Receptor binding: Tilapia leptins demonstrate bioactivity in mammalian cell systems expressing leptin receptors, though with lower potency than mammalian leptins, indicating partial conservation of receptor-binding domains .
This evolutionary divergence suggests leptin's role has been adapted to different metabolic strategies across vertebrate lineages while maintaining core functions in energy homeostasis regulation.
How can research on tilapia Leptin-B inform understanding of metabolic disorders?
Tilapia Leptin-B research offers several insights relevant to metabolic disorders:
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Leptin resistance models: Studies demonstrate that diet-induced obesity (DIO) in Nile tilapia fed high-carbohydrate or high-fat diets leads to leptin resistance (LR), similar to mammalian models . Interestingly, high-carbohydrate DIO fish retain leptin action in lipid metabolism while showing LR in glucose metabolism regulation, while high-fat DIO fish show the opposite pattern .
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Reversibility of leptin resistance: Fasting for one week completely recovers leptin actions in the regulation of both lipid and glucose metabolism in tilapia, suggesting potential therapeutic approaches .
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Selective leptin resistance: The observation that leptin resistance can differentially affect glucose versus lipid metabolism pathways provides a model for studying pathway-specific resistance mechanisms .
These insights could inform novel approaches to understanding and treating selective leptin resistance in human metabolic disorders, particularly in understanding how different dietary compositions might affect specific metabolic pathways.
What methodological challenges exist in translating findings between fish and mammalian leptin research?
Researchers face several methodological challenges:
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Dual paralog system: The presence of two functionally distinct leptin paralogs in fish requires careful experimental design to differentiate their effects, unlike the single-gene system in mammals .
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Divergent expression patterns: Different primary sites of leptin production (liver in fish versus adipose in mammals) necessitate tissue-specific approaches to studying regulation .
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Environmental influences: As ectotherms, fish leptin function is potentially influenced by environmental factors like temperature, requiring controlled experimental conditions .
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Receptor sensitivity differences: Fish leptins show lower activity in mammalian receptor systems, complicating cross-species analyses .
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Nutritional response divergence: Opposite fasting responses (increased in fish, decreased in mammals) require careful interpretation of nutritional manipulation studies .
These challenges highlight the importance of species-specific approaches while seeking conserved mechanisms that might represent fundamental aspects of vertebrate energy homeostasis regulation.
Product Science Overview
Leptin is a protein hormone primarily produced in adipose cells. It plays a crucial role in regulating energy homeostasis by inhibiting hunger, thus helping to maintain body weight. Leptin binds to nuclear receptors in the hypothalamus, particularly in the arcuate nucleus. In cases of obesity, there is often a decrease in sensitivity to leptin, leading to a failure to recognize satiety despite high levels of energy stores or leptin itself.
In tilapia (Oreochromis niloticus), two leptin sequences (tLepA and tLepB) and one leptin receptor sequence (tLepR) have been identified. The full-length cDNA of tLepR is 3423 base pairs long, encoding a protein of 1140 amino acids. This protein contains all the functionally important domains conserved among vertebrate leptin receptors. The cDNAs of tLepA and tLepB are 486 and 459 base pairs long, encoding proteins of 161 and 152 amino acids, respectively. The three-dimensional structures of tLepA and tLepB show strong conservation of tertiary structure with that of human leptin, comprising four helices .
Leptin-B Tilapia Recombinant is produced in Escherichia coli (E. coli) as a single, non-glycosylated polypeptide chain containing 152 amino acids and having a molecular mass of 15,243 Daltons. The recombinant protein is purified using proprietary chromatographic techniques .
Physical Appearance and Formulation
Leptin-B Tilapia Recombinant is available as a sterile, filtered, white lyophilized (freeze-dried) powder. It is lyophilized from a concentrated solution containing sodium bicarbonate (NaHCO3) at a 1:2 salt-to-protein ratio. For reconstitution, it is recommended to use sterile water or 0.4% NaHCO3 adjusted to pH 8-9, at a concentration of no less than 100 µg/ml, which can then be further diluted into other aqueous solutions .
Stability and Storage
The lyophilized form of Leptin-B Tilapia is stable at room temperature for up to three weeks. However, for long-term storage, it should be kept desiccated below -18°C. Upon reconstitution, it should be stored at 4°C for 2-7 days and for future use below -18°C. To ensure long-term stability, it is recommended to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA). Freeze-thaw cycles should be avoided .
Purity and Biological Activity
Leptin-B Tilapia Recombinant has a purity greater than 95%, as determined by gel filtration analysis and SDS-PAGE. The first six N-terminal amino acids of the recombinant protein are Ala-Leu-Leu-Thr-Lys-Gly. Biologically, tilapia leptins have been found to promote the proliferation of BAF/3 cells stably transfected with the long form of the human leptin receptor, although their activity is lower than that of mammalian leptin. Additionally, tilapia leptins are active in promoting STAT-LUC activation in COS7 cells transfected with the tilapia leptin receptor but not in cells transfected with the human leptin receptor. Among the tilapia leptins, Leptin A is more active than Leptin B .
