Recombinant Human Leptin

Recombinant Human Leptin is used in research applications to study and manipulate energy homeostasis, neuroendocrine function, metabolism, and related pathologies, particularly in models of leptin deficiency or metabolic disorders. Its use enables precise investigation of leptin’s physiological and therapeutic roles.

Key scientific reasons to use recombinant human leptin include:

• Modeling Leptin Deficiency and Replacement: Recombinant leptin is essential for studying the effects of leptin replacement in states of congenital or acquired leptin deficiency, such as lipodystrophy, hypothalamic amenorrhea, and certain forms of lipoatrophy. In these contexts, leptin administration has been shown to normalize neuroendocrine and metabolic abnormalities, improve insulin resistance, reduce hepatic steatosis, and correct dyslipidemia.

• Metabolic and Endocrine Research: Leptin is a master regulator of energy balance, appetite, and body adiposity. Recombinant leptin allows researchers to dissect its roles in glucose homeostasis, thermogenesis, reproductive function, and immune responses.

• Therapeutic Mechanism Studies: Recombinant leptin is used to evaluate its therapeutic potential in metabolic syndrome, insulin resistance, and weight regulation. For example, in leptin-deficient or hypoleptinemic states, leptin therapy improves insulin sensitivity, lipid profiles, and reduces central fat mass.

• Neuroendocrine and Behavioral Effects: Leptin’s actions extend to the central nervous system, influencing appetite, fertility, cognition, and mental health. Recombinant leptin is used to explore these effects in both animal models and clinical studies.

• Drug Development and Combination Therapies: Recombinant leptin serves as a tool for preclinical and clinical evaluation of combination therapies (e.g., with thiazolidinediones) and for the development of leptin sensitizers for obesity and metabolic diseases.

• Experimental Control: Using recombinant human leptin ensures batch-to-batch consistency, species specificity, and the absence of confounding factors present in serum-derived leptin, making it ideal for controlled mechanistic studies.

In summary, recombinant human leptin is a critical reagent for elucidating leptin’s physiological functions, modeling disease states, and testing therapeutic interventions in metabolic and neuroendocrine research.

Yes, recombinant human leptin can be used as a standard for quantification or calibration in ELISA assays, provided the assay is designed to recognize the recombinant form and the standard is properly characterized and traceable.

Key Points:

• Recombinant leptin as a standard: Most commercial human leptin ELISA kits use E. coli-expressed recombinant human leptin as the calibrator. This is explicitly stated for several kits (e.g., R&D Systems Quantikine, Proteintech, Eagle Biosciences, RayBiotech, MyBioSource). The dose-response curves for recombinant leptin are parallel to those of natural human leptin, allowing for accurate relative quantification.

• Traceability to WHO standard: Some kits (e.g., Eagle Biosciences, IBL-America, and others) indicate that their recombinant leptin standard is traceable to the WHO International Standard (NIBSC/WHO 97/594). This allows for conversion of results to international units (mIU/mL) if needed.

• Assay compatibility: The assay must be validated to recognize both recombinant and natural leptin. Most sandwich ELISA kits for human leptin are designed to detect both forms, as confirmed by specificity and recovery data.

• Calibration and linearity: When using recombinant leptin as a standard, ensure that the dilution series covers the expected concentration range of your samples and that the assay demonstrates linearity and recovery within that range.

Recommendations:

• Use a well-characterized recombinant human leptin standard (preferably traceable to WHO 97/594).
• Follow the manufacturer’s protocol for standard preparation and dilution.
• Validate the standard in your specific assay by assessing linearity, recovery, and parallelism with natural leptin samples if possible.

In summary, recombinant human leptin is suitable for use as a standard in ELISA assays for leptin quantification, provided the assay is compatible and the standard is properly calibrated.

Recombinant Human Leptin has been validated for several key applications in published research, primarily in the study and treatment of leptin deficiency syndromes, metabolic disorders, and neuroendocrine regulation.

Validated Applications:

• Treatment of Leptin Deficiency Syndromes:
  Recombinant human leptin has been used to treat congenital leptin deficiency, resulting in normalization of appetite, reduction of severe obesity, and restoration of neuroendocrine function, including ovulatory menstruation in women with hypothalamic amenorrhea.

• Lipodystrophy and Metabolic Syndrome:
  It has been validated for improving insulin resistance, reducing hepatic gluconeogenesis, lowering hemoglobin A1c, improving hyperlipidemia, and reversing hepatic steatosis in patients with congenital or acquired lipodystrophy.

• Weight Loss and Fat Reduction:
  Subcutaneous administration of recombinant leptin has demonstrated a dose-dependent effect on weight and fat loss in both lean and obese subjects, with fat loss accounting for the majority of weight reduction.

• Neuroendocrine Regulation:
  Leptin replacement therapy has been shown to normalize levels of estrogen, thyroid hormones, IGF-1, and restore ovulatory cycles in hypoleptinemic women.

• Improvement of Bone Metabolism:
  Studies indicate that leptin increases markers of bone formation, suggesting a role in bone metabolism, particularly in women with hypothalamic amenorrhea.

• Correction of Dyslipidemia and Hepatic Insulin Sensitivity:
  Leptin treatment has been associated with marked improvement in dyslipidemia, hepatic insulin sensitivity, and decreased visceral fat in lipodystrophic patients.

• Combination Therapy for Obesity:
  Recombinant leptin has been studied in combination with other agents (e.g., pramlintide, amylin agonists) to overcome leptin resistance and achieve synergistic fat-specific weight loss in obesity models.

• Regulation of Immune Function and Inflammation:
  Leptin is involved in the regulation of immune responses, inflammation, hematopoiesis, angiogenesis, and wound healing, with recombinant forms used to study these processes in vitro and in vivo.

Additional Research Uses:

• Mechanistic Studies:
  Recombinant leptin is widely used in basic research to elucidate its role in energy homeostasis, neuroendocrine function, metabolism, and organ fibrogenesis.

• Clinical Trials:
  Ongoing clinical trials are evaluating its effects on insulin action in type 2 diabetes mellitus and other metabolic disorders.

Summary Table:

These applications are supported by multiple clinical trials, mechanistic studies, and proof-of-concept research in both human and animal models.

To reconstitute and prepare Recombinant Human Leptin protein for cell culture experiments, dissolve the lyophilized protein in sterile, distilled water or an appropriate buffer (such as 20 mM Tris-HCl, pH 8.0 or PBS), typically to a concentration of 1.0–5.0 mg/mL. Do not vortex the solution; instead, gently mix by inversion or pipetting to avoid protein denaturation.

Step-by-step protocol:

1. Centrifuge the vial briefly before opening to collect all contents at the bottom.
2. Warm the vial to room temperature before opening to minimize condensation.
3. Add sterile, distilled water or buffer (e.g., 20 mM Tris-HCl, pH 8.0, or PBS) to achieve the desired concentration (commonly 1.0–5.0 mg/mL for stock solutions).
4. Gently mix by pipetting up and down or by slow inversion. Avoid vigorous shaking or vortexing.
5. Let the solution sit at room temperature for 15–30 minutes to ensure complete dissolution.
6. Aliquot the stock solution to minimize freeze-thaw cycles.
7. For long-term storage, dilute further in a buffer containing a carrier protein (e.g., 0.1% BSA or HSA) and store aliquots at –20°C to –80°C. For short-term use (up to 1 week), store at 2°C to 8°C.
8. Avoid repeated freeze-thaw cycles to preserve protein activity.

Preparation for cell culture:

• Before adding to cell culture, dilute the stock solution to the desired working concentration using sterile culture medium or buffer containing a carrier protein (e.g., 0.1% BSA) to prevent adsorption and loss of activity.
• If using serum-free conditions, avoid animal-derived carrier proteins and consider alternatives like trehalose.

Additional notes:

• The optimal working concentration of leptin should be determined empirically for each cell type and experimental setup.
• Confirm protein solubility and integrity by SDS-PAGE if needed.
• Endotoxin levels should be checked if sensitive cell types are used; recombinant leptin preparations typically have low endotoxin (<0.005 EU/µg).

Summary Table:

This protocol ensures maximum solubility, stability, and bioactivity of recombinant human leptin for cell culture experiments.