Leptin Ovine, MTS

What is MTS-leptin and how does it differ from standard recombinant ovine leptin?

MTS-leptin is a modified form of ovine leptin that contains a membrane translocating sequence tag composed of 10 amino acids (Val-Leu-Leu-Pro-Val-Leu-Leu-Ala-Ala-Pro) located at the N-terminus. This modification is designed to enhance the hormone's ability to cross cellular membranes and potentially the blood-brain barrier. Standard recombinant ovine leptin (roleptin) lacks this sequence. MTS-leptin produced in E.Coli is a single, non-glycosylated polypeptide chain containing 157 amino acids with a molecular mass of approximately 17.5 kDa . The sequence of the first five N-terminal amino acids has been determined to be Ala-Val-Pro-Ile-Arg . This structural modification impacts the hormone's bioavailability and potentially its experimental applications.

What are the primary functions of leptin in ovine physiology?

Leptin in sheep serves as a 16-kDa peptide hormone primarily secreted from white adipocytes, functioning as the key afferent signal from fat cells in the feedback system controlling body fat stores . It plays crucial roles in:

• Regulation of food intake and energy balance

• Transport across the blood-brain barrier via the short form leptin receptor (LeptRa)

• Modulation of reproductive function, including effects on ovarian development and folliculogenesis

• Seasonal adaptation of metabolic and reproductive systems

• Integration of nutritional status with neuroendocrine functioning

Research indicates that leptin's actions are seasonally dependent in sheep, with differential responses observed under long-day (LD) versus short-day (SD) photoperiods, particularly regarding melatonin secretion .

How does the short form of the leptin receptor (LeptRa) function in relation to MTS-leptin transport to the central nervous system?

LeptRa plays a critical role in facilitating leptin transport across the blood-brain barrier to the central nervous system (CNS). This transport mechanism is essential for leptin's central actions on hypothalamic centers controlling energy homeostasis and reproduction. Research indicates that MTS-leptin and roleptin affect LeptRa expression in specific brain regions including the arcuate nucleus (ARC), choroid plexus (ChP), and anterior pituitary (AP) .

Methodologically, researchers have observed that LeptRa mRNA transcript levels are influenced by photoperiod, being predominantly detected in the ARC and ChP during short-day photoperiods, while expression in the anterior pituitary is higher during long-day photoperiods . Importantly, LeptRa expression was not detected in the preoptic area (POA) and ventromedial/dorsomedial hypothalamus (VMH/DMH), suggesting region-specific roles of this receptor form . These findings highlight the importance of considering brain region specificity when designing experiments involving leptin transport mechanisms.

What is the relationship between VEGF signaling and leptin transport in ovine models?

Recent research has revealed an intriguing relationship between the vascular endothelial growth factor (VEGF) system and leptin transport. MTS-leptin and roleptin administration influence VEGFA and VEGFR2 concentrations in specific hypothalamic nuclei, the choroid plexus, and anterior pituitary . This relationship appears to be modulated by both photoperiod and nutritional status.

In experimental paradigms, VEGF system protein concentrations show photoperiod-dependent patterns similar to LeptRa expression. This suggests that VEGF signaling may complement or facilitate leptin transport across the blood-brain barrier, potentially through vascular remodeling or altered permeability. Researchers investigating this relationship should consider dual assessment of both leptin receptor and VEGF system components when studying leptin transport mechanisms .

What are the optimal protocols for administering MTS-leptin in ovine experimental models?

When designing experiments with MTS-leptin in sheep models, several methodological considerations are critical:

• Dosage determination: Studies have utilized various doses ranging from 0.5-1.0 μg/kg body weight for intracerebroventricular (ICV) administration . The appropriate dose depends on the specific research question and route of administration.

• Administration routes:

  • Central administration: Direct ICV infusion into the third ventricle requires surgical placement of cannulae and confirmation of CSF flow prior to experiment initiation .

  • Peripheral administration: Intravenous or subcutaneous injection may require higher doses to achieve central effects.

• Timing considerations: Administration is typically conducted with reference to photoperiod, with samples collected at standardized intervals (e.g., every 15-30 minutes) for acute response studies .

• Control considerations: Proper controls include Ringer-Locke buffer (pH 7.4) for vehicle control and potentially a dose-response design with multiple leptin concentrations .

• Reconstitution protocol: Lyophilized MTS-leptin should be reconstituted in sterile 0.02% NaHCO₃ at concentrations not less than 100 μg/ml, which can then be further diluted to appropriate concentrations .

For long-term storage stability, it is recommended to add a carrier protein (0.1% HSA or BSA) and avoid freeze-thaw cycles .

How can researchers assess leptin resistance in experimental ovine models?

Leptin resistance assessment in sheep requires multiple methodological approaches:

• Physiological markers: In overweight animals, physical appearance provides initial indication; persistent appetite and carbohydrate cravings, especially at night, suggest leptin resistance .

• Biochemical assessment:

  • Blood tests for elevated reverse T3 levels, which typically accompany leptin resistance

  • Salivary cortisol measurements, with higher levels later in the day indicating leptin resistance

  • Direct measurement of circulating leptin levels, which are typically elevated in leptin-resistant states

• Response testing:

  • Challenge tests using standardized MTS-leptin or roleptin doses with measurement of expected physiological responses

  • Assessment of hypothalamic gene expression changes following leptin administration

  • Evaluation of feeding behavior modifications following leptin treatment

• Tissue-specific analyses:

  • Quantification of hypothalamic LeptRa expression

  • Assessment of intracellular signaling pathway activation (e.g., STAT3 phosphorylation)

  • Examination of inflammatory markers that may contribute to leptin resistance

Research indicates that both photoperiodic and nutritional signals influence leptin transport to the CNS via LeptRa, suggesting that experimental designs should account for both seasonality and nutritional status .

How does photoperiod influence the efficacy of MTS-leptin in ovine models?

Photoperiod exerts profound effects on MTS-leptin efficacy in sheep, with experimental evidence demonstrating season-dependent actions:

• Differential receptor expression: LeptRa mRNA transcript levels show seasonal variation, with predominant detection in the arcuate nucleus and choroid plexus during short-day photoperiods, while anterior pituitary expression is higher during long-day photoperiods .

• Melatonin interactions: Leptin's effects on melatonin secretion show photoperiodic dependence. In vitro studies demonstrate that recombinant ovine leptin negatively modulates pineal gland melatonin secretion during long days but stimulates secretion during short days . In vivo ICV administration of roleptin confirms this biphasic effect, with dose-dependent increases in melatonin during short days and decreases during long days .

• Prolactin regulation: Unlike melatonin, plasma concentrations of prolactin are greater during long days than short days, but leptin administration decreases prolactin levels regardless of photoperiod .

These findings highlight the critical importance of controlling for and documenting photoperiod conditions in experimental protocols involving MTS-leptin. Researchers should standardize the photoperiodic conditions and consider seasonal timing when designing experiments and interpreting results.

What is the relationship between nutritional status and MTS-leptin/roleptin efficacy in ovine models?

Nutritional status significantly influences MTS-leptin and roleptin efficacy through multiple mechanisms:

• Acute fasting effects: In research protocols, 72-hour fasting periods alter the expression of LeptRa and VEGF system components, modifying leptin transport efficacy .

• Long-term adiposity influence: Experimental models creating lean or fat sheep through 5-month dietary interventions demonstrate that changes in adiposity influence leptin transport mechanisms . Specifically, adiposity alterations affect how adipokines like resistin impact the expression of LeptRa and VEGF system protein concentrations.

• Leptin resistance development: Long-term alterations in nutritional status can induce adaptive or pathological leptin resistance phenomena in sheep. Research indicates that resistin may be another adipokine involved in these adaptive mechanisms .

When designing experiments involving MTS-leptin, researchers should:

• Document and standardize nutritional status of experimental animals

• Consider both acute fasting and long-term adiposity as experimental variables

• Potentially measure multiple adipokines (not just leptin) to understand network effects

• Account for adaptive changes in leptin sensitivity when interpreting results from long-term studies

How does MTS-leptin influence ovarian function and follicular development in sheep?

MTS-leptin exerts significant effects on ovarian function through direct and indirect mechanisms:

• Expression patterns: Immunohistochemical analyses reveal that both leptin and its receptor (LEPR) are expressed in ovine ovaries, establishing the foundation for direct ovarian effects .

• In vitro follicular development: Research demonstrates that leptin supplementation at appropriate concentrations enhances secondary follicle development in sheep. Specifically, 25 ng/mL leptin supplementation significantly increases:

  • Antrum formation rates

  • Development of fully grown oocytes

  • Glutathione (GSH) levels in oocytes

  • Mitochondrial activity in follicular cells

• Maturation outcomes: Leptin supplementation at 25 ng/mL promotes higher in vitro maturation rates, with a higher percentage of oocytes reaching metaphase II (MII) compared to control medium .

These findings suggest that MTS-leptin could serve as a valuable supplement in ovarian tissue culture systems and in vitro maturation protocols. The optimal concentration appears to be 25 ng/mL, with evidence showing improved follicular development, GSH levels, and mitochondrial activity compared to control conditions .

What role does the leptin system play in pubertal development in sheep compared to other species?

The leptin system's role in pubertal development shows important species-specific variations:

• Permissive factor: Across species, leptin acts primarily as a permissive rather than triggering factor for puberty onset. It signals energy sufficiency required for reproductive maturation .

• Species differences: While leptin administration can advance puberty timing in rodents at low doses insufficient to alter metabolism, effects in non-human primates are less clear and require very high doses to sustain LH levels in fasted monkeys .

• Seasonal breeders: Sheep and other seasonal breeders have evolved divergent strategies favoring photoperiodic cues over metabolic signals for reproductive timing . This represents a critical species difference from rodent and primate models.

• Energy balance signaling: Across species, leptin prevents delayed pubertal development caused by negative energy balance, functioning as a metabolic signal of energy sufficiency .

• Early developmental importance: Research with leptin antagonists demonstrates a previously unrecognized role of leptin in postnatal maturation of reproductive organs, including ovaries with marked impact on primordial follicle development .

For researchers studying puberty in sheep models, these species differences highlight the importance of considering photoperiodic influences alongside metabolic signals, differentiating between permissive versus triggering factors, and recognizing the complex integration of multiple endocrine and environmental signals.

What are the optimal storage and reconstitution protocols for MTS-leptin to maintain biological activity?

Proper handling of MTS-leptin is critical for experimental reproducibility. The recommended protocols include:

• Storage conditions:

  • Lyophilized MTS-leptin, while stable at room temperature for up to 3 weeks, should be stored desiccated below -18°C for long-term stability

  • Upon reconstitution, store at 4°C for short-term use (2-7 days)

  • For future use, store below -18°C

  • For long-term storage, add a carrier protein (0.1% HSA or BSA) to enhance stability

  • Avoid repeated freeze-thaw cycles to maintain biological activity

• Reconstitution protocol:

  • Reconstitute lyophilized MTS-leptin in sterile 0.02% NaHCO₃

  • Maintain concentration at not less than 100 μg/ml

  • This stock can be further diluted to other aqueous solutions as needed for specific experimental applications

• Activity verification:

  • Biological activity can be verified by inducing proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor

  • Purity assessment via SEC-HPLC and SDS-PAGE should show greater than 98% purity

These handling protocols are essential for maintaining the structural integrity and biological activity of MTS-leptin in research applications.

What tools and approaches are available for temporarily blocking leptin action in ovine models?

Recent advances have developed several tools for temporary/reversible blockade of leptin action, offering advantages over permanent genetic models:

• Leptin antagonists: Synthetic compounds that bind to leptin receptors without activating downstream signaling pathways, effectively blocking endogenous leptin action .

• Antibodies against leptin binding domain: These target the leptin binding domain of leptin receptors (LepR), preventing leptin from interacting with its receptor .

• Application insights:

  • Treatment of rats with leptin antagonist during early postnatal life impairs the appropriate development of several organs, including marked decrease of ovarian primordial follicles

  • This approach has revealed previously unrecognized roles of leptin in postnatal maturation of reproductive organs

  • The reversible nature allows for temporal studies of leptin action during specific developmental windows

• Experimental considerations:

  • Dose-response relationships should be established

  • Timing of administration is critical for developmental studies

  • Verification of blockade effectiveness through measurement of downstream signaling pathways

  • Potential for tissue-specific delivery to target particular organ systems

These tools represent significant methodological advances for investigators seeking to understand leptin's physiological roles without the confounding developmental abnormalities present in genetic knockout models.