Leptin-A Tilapia

What is Leptin-A in tilapia and how does it differ from mammalian leptin?

Leptin-A (LepA) is one of two leptin isoforms in tilapia (Oreochromis niloticus), with LepA being the dominant ortholog. The full-length cDNA of tilapia LepA (tLepA) is 486 bp, encoding a protein of 161 amino acids. Despite relatively low primary sequence homology with mammalian leptin, the three-dimensional structure of tLepA demonstrates strong conservation of tertiary structure, comprising 4 helices similar to human leptin . Tilapia has two leptin genes (lepA and lepB), whereas mammals typically have only one. This reflects the whole genome duplication event in teleost evolution. LepA appears to have retained most of the ancestral functions related to energy homeostasis in fish .

Where is Leptin-A primarily expressed in tilapia?

Unlike mammals where leptin is predominantly expressed in adipose tissue, tilapia LepA demonstrates high expression in both the liver and subcutaneous adipose tissue (SCAT) . Studies have verified comparable high expression levels of leptin in SCAT as in liver in Nile tilapia . This dual-site expression pattern differs from mammals and may reflect evolutionary adaptations in the regulation of energy metabolism in fish, which store lipids in multiple tissues including liver, muscle, and dispersed adipose deposits .

What are the basic physiological functions of Leptin-A in tilapia?

Leptin-A in tilapia serves several fundamental functions:

• Appetite regulation: Intraperitoneal injection of recombinant tilapia LepA (NtrelepA) significantly reduces cumulative food intake 1-7 hours after treatment, confirming an acute anorexic effect similar to mammals .

• Energy metabolism: LepA activates catabolism and promotes energy utilization in tilapia, particularly during periods of altered nutritional state .

• Growth regulation: LepA interacts with the growth hormone (GH)/insulin-like growth factors (IGFs) axis. Recombinant tilapia LepA increases hepatic gene expression of igf-1, igf-2, ghr-1, and ghr-2 in isolated hepatocytes .

• Glucose and lipid metabolism: LepA participates in regulating both glucose and lipid metabolism, though with selective actions depending on nutritional status .

How is recombinant tilapia Leptin-A produced for research purposes?

Recombinant tilapia Leptin-A (rtLepA) is typically produced using an E. coli expression system. The process involves:

• Obtaining the fragment encoding the mature protein of Nile tilapia Leptin A (Ala21-Cys161) with 6 tandem histidine residues at the N-terminal.

• Digesting the fragment using restriction enzymes (NdeI and XhoI) and cloning it into an expression vector such as pET-32a (+).

• Transforming the recombinant plasmid into E. coli competent cells (e.g., TransB (DE3)) using the heat shock method .

Commercial recombinant Leptin-A tilapia is available as a single, non-glycosylated polypeptide chain containing 161 amino acids with a molecular mass of 16,491 Dalton, purified using chromatographic techniques. For research applications, the lyophilized protein is typically reconstituted in sterile water or 0.4% NaHCO₃ adjusted to pH 8-9, with recommended concentrations not less than 100μg/ml .

How is Leptin-A regulated by different metabolic states in tilapia?

The regulation of Leptin-A in tilapia shows interesting patterns that sometimes differ from mammals:

• Fasting response: Circulating LepA levels and both lepa and lepr gene expression increase after 3-weeks of fasting and decline to control levels 10 days following refeeding . This pattern is opposite to mammals, where leptin levels typically decrease during fasting.

• Diet-induced obesity: In diet-induced obesity (DIO) models, leptin resistance develops but with selective effects depending on the type of diet. High-carbohydrate diet (HCD) fish retain leptin action in lipid metabolism but show resistance in glucose metabolism regulation, while high-fat diet (HFD) fish show the reverse pattern .

• Recovery from diet-induced resistance: Fasting DIO tilapia for 1 week completely recovers leptin actions in both lipid and glucose metabolism regulation, suggesting a reversible adaptive mechanism .

• Dietary lipid effects: Increased dietary lipid supplementation significantly raises serum leptin levels in genetically improved farmed tilapia (GIFT) juveniles compared to control diets. The optimal dietary lipid level for specific growth rate (SGR) and feed conversion ratio (FCR) has been determined to be approximately 10.5% .

This metabolic regulation pattern suggests leptin in tilapia may function as an indicator of energy reserves rather than simply a satiety signal as in mammals .

What evidence exists for a leptin-insulin axis in tilapia, and how does it compare to mammals?

Recent research provides evidence for a leptin-insulin axis in tilapia that shares functional similarities with the mammalian "adipoinsular axis" but with some important distinctions:

This bidirectional relationship provides important insights into the evolution of metabolic regulation in vertebrates .

What mechanisms underlie leptin resistance in diet-induced obesity models in tilapia?

Leptin resistance (LR) in diet-induced obesity (DIO) tilapia exhibits several intriguing characteristics:

• Selective resistance: Unlike mammals where leptin resistance often affects multiple metabolic pathways, tilapia show selective leptin resistance depending on diet type:

  • High-carbohydrate diet (HCD) fish maintain leptin sensitivity in lipid metabolism but develop resistance in glucose metabolism

  • High-fat diet (HFD) fish show the opposite pattern, maintaining sensitivity in glucose metabolism but developing resistance in lipid metabolism

• Tissue-specific effects: The selective nature of leptin resistance suggests tissue-specific alterations in leptin signaling pathways, particularly in liver and adipose tissues .

• Reversibility: A key finding is that fasting DIO tilapia for just one week completely recovers leptin action in both lipid and glucose metabolism regulation, indicating that the resistance mechanisms are highly adaptable and reversible .

• Evolutionary significance: This selective regulation pattern may represent an adaptive mechanism that evolved to maximize energy storage when different nutrient types are abundant, allowing fish to store surplus calories efficiently regardless of whether they come from carbohydrates or fats .

These findings suggest that leptin may retain more activities in animals with leptin resistance than previously believed, challenging simplistic models of complete leptin signaling impairment in obesity .

How does Leptin-A interact with the GH/IGF growth regulatory axis in tilapia?

Leptin-A in tilapia demonstrates significant interactions with the growth hormone (GH)/insulin-like growth factors (IGFs) axis:

• Divergent regulation patterns: The pattern of leptin regulation by metabolic state in tilapia is similar to that observed for pituitary GH but opposite to that of hepatic GH receptor (GHR) and/or IGF dynamics. This suggests coordinated but distinct regulatory mechanisms governing these two systems .

• Direct stimulation of growth factors: Recombinant tilapia LepA (rtLepA) directly increases hepatic gene expression of igf-1, igf-2, ghr-1, and ghr-2 in isolated hepatocytes following 24-hour incubation. This indicates leptin can directly modulate the expression of key components of the growth axis at the hepatic level .

• Nutritional state coordination: The complementary patterns of regulation during fasting and refeeding suggest leptin and GH/IGF systems work together to coordinate energy allocation between metabolism and growth, optimizing survival during periods of nutritional stress .

• Evolutionary conservation: These interactions appear to represent evolutionarily conserved mechanisms for coordinating energy status with growth potential, though the specific patterns may differ between fish and mammals .

This leptin-GH/IGF interaction provides a mechanism for tilapia to balance energy expenditure between immediate metabolic needs and long-term growth potential based on nutritional status .

What methodological approaches are most effective for studying leptin function in tilapia?

Several complementary methodological approaches have proven effective for investigating leptin function in tilapia:

In vitro methods:

• Isolated hepatocyte cultures: Effective for studying direct effects of recombinant leptin on gene expression of growth factors, receptors, and metabolic enzymes .

• Isolated Brockmann bodies (pancreatic islets): Useful for examining leptin's effects on insulin and glucagon expression and secretion under controlled glucose concentrations .

In vivo methods:

• Intraperitoneal (i.p.) injections: Administration of recombinant tilapia LepA (400 ng/g BW) has been effectively used to study acute effects on food intake and metabolism .

• Dietary manipulation models: Creating high-carbohydrate diet (HCD) or high-fat diet (HFD) models to study diet-induced obesity and leptin resistance .

• Fasting-refeeding protocols: Using controlled fasting periods (3 weeks) followed by refeeding to study dynamic regulation of leptin and associated pathways .

Molecular techniques:

• Gene expression analysis: Quantitative PCR to measure expression of lepa, lepb, and lepr in various tissues .

• Protein quantification: ELISA or other immunological methods to measure circulating leptin levels .

• Metabolic parameters assessment: Measuring growth parameters (SGR, FCR), nucleic acids, and metabolic indicators to correlate with leptin function .

The combination of both in vitro and in vivo approaches, coupled with molecular analyses, provides the most comprehensive understanding of leptin function in tilapia .

How can tilapia leptin research inform comparative endocrinology across vertebrates?

Tilapia leptin research offers valuable insights for comparative endocrinology:

These comparative aspects highlight how tilapia serves as an excellent model for understanding the evolution of energy homeostasis mechanisms across vertebrates .

What are the technical challenges in designing experiments to study leptin signaling in tilapia?

Researchers face several technical challenges when studying leptin signaling in tilapia:

• Species-specific reagents: The limited availability of species-specific antibodies and assay reagents often necessitates developing custom tools or validating cross-reactivity with commercially available options. Using recombinant tilapia leptin rather than mammalian leptin is crucial for physiologically relevant results .

• Stability and handling of recombinant protein: Recombinant tilapia Leptin-A requires careful handling, with specific reconstitution protocols using 0.4% NaHCO₃ adjusted to pH 8-9. Even with proper handling, lyophilized Leptin-A remains stable at room temperature for only about 3 weeks and reconstituted protein should be stored at 4°C for short periods (2-7 days) or below -18°C for longer storage .

• Tissue sampling limitations: The small size of specific tissues (like Brockmann bodies) and the dispersed nature of adipose tissue in fish present challenges for obtaining sufficient material for analysis .

• Diverse lipid storage sites: Unlike mammals where adipose tissue is the primary lipid storage site, tilapia store lipids in multiple tissues including liver, muscle, and dispersed adipose deposits, complicating the study of lipid metabolism .

• Diet formulation and feeding regimens: Creating standardized experimental diets with precise nutrient compositions is critical for studies on diet-induced obesity and leptin resistance but requires specialized expertise in fish nutrition .

Addressing these challenges requires careful experimental design and often the development of custom methodologies tailored to tilapia physiology .

How should researchers interpret contradictory data on leptin function between different fish species and mammals?

When encountering contradictory data between fish species and mammals, researchers should consider:

• Evolutionary context: Teleost fish and mammals diverged approximately 450 million years ago, with teleosts undergoing an additional whole-genome duplication event. This resulted in two leptin paralogs (LepA and LepB) in most fish species compared to a single leptin gene in mammals. These evolutionary differences may underlie functional divergence .

• Environmental adaptations: Fish inhabit aquatic environments with different energetic challenges than terrestrial mammals, potentially leading to fundamentally different metabolic strategies. For instance, the opposite regulation of leptin during fasting between fish and mammals may reflect different strategies for energy mobilization in these environments .

• Methodological considerations: Contradictions may arise from:

  • Use of heterologous (mammalian) leptin in fish studies rather than species-specific recombinant protein

  • Different sampling times or physiological states

  • Variations in experimental conditions (temperature, feeding status, etc.)

• Selective pressures on energy storage: Fish typically experience more variable food availability in natural environments than laboratory mammals, potentially selecting for different leptin signaling strategies .

• Data integration approach: Rather than viewing contradictions as experimental failures, researchers should use them to develop more nuanced models of leptin function that incorporate evolutionary divergence and convergence across vertebrate lineages .

This interpretive framework allows researchers to reconcile apparently contradictory findings and develop more comprehensive models of leptin function across vertebrates .

What control measures should be implemented when studying leptin's effects on metabolism in tilapia?

Critical control measures for leptin-metabolism studies in tilapia include:

• Baseline physiological parameters:

  • Establish normal ranges for growth parameters (SGR, FCR, HSI) in your specific tilapia strain and age group

  • Document baseline leptin levels and gene expression patterns across relevant tissues

  • Control for diurnal variations by consistent sampling times

• Nutritional status standardization:

  • Implement acclimation periods with controlled diets before experiments (typically 2-4 weeks)

  • For fasting studies, standardize fasting duration (3 weeks has been effective in previous studies)

  • For refeeding studies, control feeding amounts and timing precisely

• Appropriate controls for recombinant protein administration:

  • Include vehicle controls (e.g., the buffer used for reconstitution of leptin)

  • Use heat-inactivated leptin as an additional control to verify specific bioactivity

  • Include dose-response designs rather than single-dose administration

• Environmental parameters:

  • Maintain consistent water temperature, pH, dissolved oxygen, and photoperiod

  • House experimental and control groups in identical tank systems with equal stocking densities

  • Randomize tank assignments to control for potential tank effects

• Methodology validation:

  • Verify the bioactivity of recombinant leptin batches before use

  • Include positive controls (e.g., known leptin-responsive genes) in expression studies

  • Validate all primers and antibodies for specificity in tilapia tissues

These control measures strengthen experimental rigor and increase the reliability of data on leptin's metabolic effects in tilapia .

What dietary formulations are most appropriate for studying leptin function in different nutritional contexts?

Optimal dietary formulations for studying leptin function in tilapia vary by research context:

Standard/Control Diets:

• Balanced formulation: Iso-nitrogenous diets with approximately 34% dietary protein and moderate lipid levels (3-6%) represent appropriate baseline diets .

• Low-lipid control: For lipid supplementation studies, a baseline diet containing minimal lipid (0.35%) without additional lipid supplementation serves as an effective control .

High-Carbohydrate Diet (HCD) for DIO Models:

• These diets typically feature elevated digestible carbohydrates while maintaining protein content

• Useful for studying selective leptin resistance in glucose metabolism

High-Fat Diet (HFD) for DIO Models:

• Diets supplemented with fish oil to reach lipid levels of 9-15% while maintaining iso-nitrogenous formulation

• Effective for studying leptin resistance in lipid metabolism

Graded Lipid Series:
For dose-response studies, a series of diets with incrementally increasing lipid levels provides valuable insights:

• Control diet (0.35% lipid)

• Incrementally increased levels (e.g., 3.35%, 6.35%, 9.35%, 12.35%, and 15.35% lipid)

• Based on second-order polynomial regression analysis, optimal dietary lipid levels of approximately 10.5% produce optimal growth performance

Diet Preparation Considerations:

• Use high-quality ingredients with consistent composition

• Ensure homogeneous mixing of supplements

• Properly store diets to prevent oxidation of lipids

• Analyze final diets to confirm actual nutrient composition

These formulations provide appropriate experimental frameworks for investigating leptin's role across different nutritional contexts in tilapia .

How can researchers effectively measure and interpret changes in leptin signaling pathways in tilapia?

Effective measurement and interpretation of leptin signaling in tilapia requires multi-level analysis:

Transcriptional Analysis:

• Gene expression profiling: Quantitative PCR for key components:

  • Leptin system genes: lepa, lepb, lepr

  • Downstream targets: igf-1, igf-2, ghr-1, ghr-2

  • Metabolic enzymes: stearoyl CoA desaturase-1, lipoprotein lipase

  • Insulin pathway components: insa, glub

• Tissue selection is critical:

  • Liver: Primary site of leptin production in tilapia

  • Subcutaneous adipose tissue: Secondary production site

  • Hypothalamic lateral tuberal nucleus: Fish homolog of arcuate nucleus

  • Brockmann bodies: Pancreatic endocrine tissue in fish

Protein Level Assessment:

• Circulating leptin: Immunological assays calibrated for tilapia leptin

• Receptor expression: Analysis of membrane fractions for leptin receptor proteins

• Post-translational modifications: Phosphorylation status of key signaling components

Functional Outputs:

• Metabolic parameters:

  • Glucose tolerance tests

  • Hepatic glycogen content

  • Lipid profiles and tissue lipid content

  • Lipolysis and lipogenesis rates

• Growth metrics:

  • Specific growth rate (SGR)

  • Feed conversion ratio (FCR)

  • Hepatosomatic index (HSI)

Integrated Data Interpretation:

• Time-course analysis: Distinguish between acute (1-7 hours) and chronic (days to weeks) effects of leptin

• Context-dependent effects: Interpret data within specific nutritional contexts (fasting, refeeding, DIO models)

• Comparative approach: Reference findings against established patterns in mammals to identify conserved versus divergent mechanisms

• Statistical modeling: Use appropriate models for complex interactions:

  • Second-order polynomial regression for dose-response relationships

  • Multivariate analysis for correlating leptin signaling with multiple physiological outcomes

This comprehensive approach allows researchers to effectively track signaling pathway changes and interpret them within relevant physiological contexts .

What are the key growth and metabolic parameters affected by leptin in tilapia?

Table 1: Effect of Different Dietary Lipid Levels on Growth Parameters and Leptin in GIFT Tilapia Juveniles

Data compiled from search result , showing significant effects of dietary lipid supplementation on growth, body composition, and hormone parameters compared to control diet. SGR = specific growth rate; FCR = feed conversion ratio; HSI = hepatosomatic index; LepR = leptin receptor; AdipoRs = adiponectin receptors.

Table 2: Comparative Effects of Fasting and Refeeding on Leptin and Related Hormones in Tilapia

Data compiled from search result , showing the pattern of leptin regulation by metabolic state is similar to that observed for pituitary GH but opposite to that of hepatic GHR and IGF dynamics in tilapia. GH = growth hormone; GHR = growth hormone receptor; IGFs = insulin-like growth factors.

This data demonstrates that leptin significantly impacts growth performance, body composition, and metabolic regulation in tilapia, with its effects being strongly influenced by nutritional status .

How does leptin resistance manifest differently in HCD-DIO versus HFD-DIO tilapia models?

Table 3: Comparison of Selective Leptin Resistance in Different Diet-Induced Obesity Models in Tilapia

Data compiled from search results , demonstrating the selective nature of leptin resistance in different DIO models and the complete recovery after fasting. HCD = high-carbohydrate diet; HFD = high-fat diet; DIO = diet-induced obesity; SCAT = subcutaneous adipose tissue.

This table highlights the remarkable finding that leptin resistance in tilapia is nutrient-specific rather than systemic, with HCD-DIO fish maintaining leptin sensitivity in lipid metabolism while developing resistance in glucose metabolism regulation, and HFD-DIO fish showing the reverse pattern . This selective regulation represents a potential evolutionary adaptation allowing fish to store surplus calories efficiently regardless of whether they come from carbohydrates or fats .

What is the evidence for a leptin-insulin axis in tilapia and how does it compare to the mammalian model?

Table 4: Comparative Analysis of Leptin-Insulin Axis in Tilapia and Mammals

Data compiled from search result , showing evidence for a leptin-insulin axis in tilapia that shares functional similarities with mammals but exhibits more dynamic and flexible relationships. insa = insulin gene a; glub = glucagon gene b.

This comparative analysis reveals that while a leptin-insulin regulatory axis exists in both tilapia and mammals, the teleost version appears more dynamic and adaptable, likely reflecting the different metabolic demands and energy storage strategies between aquatic ectotherms and terrestrial endotherms . The conservation of this axis across such evolutionary distance (approximately 450 million years) suggests it represents a fundamental vertebrate mechanism for coordinating energy homeostasis .