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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Multidimensional Characterization and Adaptive Differentiation of Leptin Gene in Eothenomys miletus: Based on Bioinformatics and System Evolution Analysis

H
Huairan Wang1
0009-0004-3687-4081
W
Wenrong Gao2
https://orcid.org/0000-0001-9521-7613
W
Wanlong Zhu1,*
0000-0001-8261-4089
1School of Life Sciences, Yunnan Normal University, Kunming, 650500, China.
2School of Biological Resources and Food Engineering, Qujing Normal University, Qujing, 655011, China.

ABSTRACT

Background: To investigate the differences between leptin gene in Eothenomys Miletus and other 55 mammal species, this study conducted a comparative analysis of multidimensional characteristics and adaptive differentiation of leptin genes.

Methods: In this study, molecular biology experimental strategies have been used to extract and reverse transcription, gene cloning and sequencing of E. miletus and bioinformatics evaluation techniques have been used to analyze the sequence and structure, multi-sequence alignment and evolutionary evaluation of E. miletus and ecological and distribution statistical strategies had been used to analyze the leptin genes of 56 mammalian species such as E. miletus.

Result: Leptin gene in E. miletus contained a Sec/SPI signal peptide, with α-helices dominating its structure. A model was constructed using leptin-LepR trimer (8 × 80.1. C leptin) as template. It encodes 127 amino acids with high proportions of leucine and serine; its molecular formula is C824H1353N221O252S8 (2658 atoms) with molecular weight of 18644.62 and theoretical isoelectric point of 5.86. The protein is unstable and hydrophilic, yet more hydrophobic than Tupaia chinensis and Homo sapiens. E. miletus was genetically close to Microtus, distant from Lagomorpha and clusters with temperate Rodentia. Its codon preference differed from Lagomorpha, with moderate GC content, GC3 content and ENC values. Motifs 1-5 were conserved in its leptin. Functional annotation shows regions related to signal peptide, leptin and extracellular domains. The ω value of ~0.5661 suggests evolutionary conservation. These results provide basis for exploring leptin gene evolution in E. miletus and mammals, offering perspective on mammalian adaptation to extreme environments and high-altitude protection.

INTRODUCTION

Leptin, a conserved protein hormone encoded by the obesity gene and primarily secreted by white adipose tissue (Priyadarshini et al., 2015), was identified in humans and mice in 1994 and has since become a key research focus. As a core regulator of energy metabolism, it modulates energy balance and suppresses food intake (Zhang et al., 2009; Sun, 2019), while also participating in immune responses (Wei et al., 2013), infectious diseases and reproduction (Jin et al., 2021; Zhang, 2015). Molecular and genetic studies have explored leptin genes in animals (Pang et al., 2016; Li et al., 2021; Meena et al., 2017), their associations with diseases (Yan et al., 2023; Ma, 2012) and leptin receptor genes (Yan et al., 2011; Li et al., 2011). Its physiological roles, linked to gene structural and functional variations, are critical for thermoregulation and metabolic heat production under environmental temperature stress. 
       
Eothenomys miletus, a Cricetidae rodent, which was endemic to the Hengduan Mountains of Yunnan (Han et al., 2021), it has been studied by our team for adaptive strategies, including physiological ecology, such as correlations between serum leptin levels and physiological parameters under varying conditions (Hou et al., 2020; Yang et al., 2013; Yang et al., 2016) and leptin-mediated regulation of body mass and energy metabolism via hypothalamic neuropeptides (Zhu, 2015). However, research on the molecular evolutionary mechanisms of its leptin gene remains limited. To clarify the leptin gene’s bioinformatics characteristics and evolutionary selection pressure, this study integrated leptin sequences from 55 mammals across five orders (Rodentia, Dermoptera, Lagomorpha, Scandentia, Primates) - taxa with wide distributions from equatorial tropics to cold zones, subject to strong temperature selection pressure. Recent studies note cold-dwelling Vulpes lagopus exhibits leptin receptor sensitivity, while tropical Tupaia belangeri has unique leptin expression regulation (Zhu et al., 2014), indicating temperature-driven adaptive evolution of leptin. However, the universality of such latitude- and temperature-related adaptations across taxa and cross-order evolutionary patterns of leptin genes, remain unclear. 
       
This study analyzed the leptin gene’s protein structure and physicochemical properties in E. miletus and performed cross-order comparative analyses of leptin sequences from 56 mammals (including E. miletus) to reveal the gene’s molecular characteristics, adaptive evolutionary signals and cross-order evolutionary patterns in mammals.

MATERIALS AND METHODS

Obtaining the complete sequence of leptin gene from E. miletus
 
From 2024 to June 2025, experiments and follow-up data analysis is conducted at the School of Life Sciences of Yunnan Normal University.
       
Primers were designed according to the sequence of leptin gene in E. miletus. The upstream primer F was 5'- ATGTGCTGGGAGACTCCTGTG-3' and the downstream primer R was 3'- CAGCATCAGCTAAGGTCC-5'. Primers were synthesized by the company. The size of the amplified product was 503bp and the temperature of cleavage was 89.8oC. The steps and operations of extracting and purifying total RNA from hypothalamus in E. miletus was details in Zhu’s (2015). The whole sequence is transmitted to NCBI (GenBank No: pv838002).
 
Sequence analysis of leptin gene in E. miletus
 
Signal peptide prediction and protein secondary and tertiary structure analysis
 
The sign peptide of leptin protein was once estimated by using SignalP-6.0 on-line tool, the secondary shape of leptin protein was once envisioned through SOPMA on line software and the tertiary shape of leptin protein used to be analyzed by means of SWISS-MODEL on line software.
 
Analysis of physicochemical properties of leptin protein from E. miletus
 
The hydrophilicity and hydrophobicity of leptin protein in E. miletus, Ochotona princeps, Tupaia belangeri and Homo sapiens were predicted and compared by using the ProtScale Program in the online software of ExPASY. The physical and chemical properties of rat leptin protein were analyzed by using the ProtParam program of ExPASY online software.
 
Cross order comparison of leptin gene sequences in 56 species
 
Collation sequence
 
Leptin gene sequences from 55 mammalian species available on the NCBI platform, along with the newly obtained sequence from E. miletus, were organized (Table 1). These sequences are sorted out by means of techniques such as (Mao et al., 2022; Yang, 1997).

Table 1: Basic information of 56 species of mammals and NCBI entry numbers.



Construction of phylogenetic tree
 
The systematic tree was constructed by the neighbor-joining (NJ) method and then the optimal model and the maximum likelihood method (ML) were used to construct the systematic tree and the self-expansion test (Bootstrap) was set to set 1000 iterations. Compare tree structures from different methods to ensure accuracy. Beautify the phylogenetic tree using Chiplot online software.
 
Distribution analysis of 56 species in five temperature zones
 
From the perspective of ecological and biological distribution, the main distribution of species is stable survival and reproduction, with large population, high density and wide distribution range. If the species are mainly distributed in a certain temperature zone, it is recorded as 1 and other distributions are recorded as 0. With the support of animal geographical distribution, ecological habits, professional literature and database, the main distribution of 56 species in 5 temperature zones were counted by Excel. Chiplot was used to analyze the clustering of 56 species in each temperature zone.
 
Codon preference and determination of GC content
 
The number of effective codons (ENC), GC content and third GC content (GC3s) of the sequence were calculated using the online tool EMBOSS Explorer.
 
Conservative region determination and functional annotation analysis of multiple sequences
 
MEME online software is used to predict the conservative motifs of multiple sequences, compare the predicted conservative motifs with Pfam, PROSITE and other databases, annotate the functional domains corresponding to the motifs and visualize the conservative regions through TBtools - II.
 
Select pressure detection
 
PAML software was used to calculate the ratio of nonsynonymous replacement rate (dN) and synonymous replacement rate (dS) (ω=dN/dS) to measure the selection pressure of leptin gene in the process of evolution. The single ratio (M0) and free ratio (M1) branching models that rely on likelihood ratio test (LRT) in Codeml program are used to detect the overall selection pressure of leptin gene and whether there is heterogeneity of selection pressure in different evolutionary branches of leptin gene.

RESULTS AND DISCUSSION

Bioinformatics analysis of leptin in E. miletus
 
Signal peptide assay
 
It showed that there was a highly reliable Sec/SPI type signal peptide in the leptin protein of E. miletus (Fig 1). The probability distribution of different regions of signal peptide is high and the probability of non-signal peptide region is low, which is consistent with the judgment of signal peptide. Cleavage site: It is predicted to be between the 21st and 22nd amino acids, with a probability of up to 0.978790. It has high reliability and the cleavage site is clear, which supports the functional localization of its secreted protein. Signal peptide type: it belongs to Sec/SPI signal peptide and the probability of signal peptide is 0.9998.

Fig 1: Prediction results of leptin signal peptide in the E. miletus.


 
Protein secondary and tertiary structure prediction
 
protein secondary structure prediction results
 
After removal of the signal peptide sequence, the mature leptin protein of E. miletus consists of 146 amino acid residues. Secondary structure prediction using SOPMA (Fig 2) revealed a predominance of α-helices (h), comprising 94 residues and accounting for 64.38% of the structure (Fig 3). Random coils (c) were the secondary major structural element, with 50 residues, while the proportion of extended strands (e) was minimal at only 1.37%.

Fig 2: The distribution of α-spiral (blue vertical line), random curl (orange short bar) and extended strand (purple vertical line) in the sequence.



Fig 3: Prediction results of secondary structure of leptin protein in E. miletus.


 
Prediction results of protein tertiary structure
 
SWISS-MODEL was used to construct the tertiary structure model of leptin protein in E. miletus (Fig 4). The core template: leptin LEPR trimer structure (8  × 80.1. C leptin) was selected to establish the model. The sequence consistency was as high as 82.04%, the global model quality score (GMQE) was 0.76 and qmeandico global was 0.76±0.07, indicating that the quality of the model was good and the construction was reasonable. The spiral structure is prominent in the modeling and the model presents a spatial folding shape dominated by α - helix, with less random curl.

Fig 4: Three-dimensional structure prediction result of leptin protein in E. miletus.


 
Determination and analysis of physical and chemical properties
 
The analysis results of physical and chemical properties of rat leptin protein showed that the number of amino acids was 127, including 20 essential amino acids, of which leucine accounted for the highest proportion and serine accounted for the second highest proportion (Table 2). Its molecular formula is C824H1353N221O252S8, with a total atomic number of 2658 and a molecular weight of 18644.62. The theoretical isoelectric point of protein is 5.86, which is acidic. The theoretical half-life of E. miletus leptin protein in vitro (mammalian reticulocytes) was 30 h, in yeast > 20 h and in Escherichia coli > 10 h. The aliphatic index of the protein was 119.04; Average hydrophilicity (GRAVY): 0.113, instability index: 50.89, which is an unstable hydrophilic protein.

Table 2: Amino acid composition of leptin protein in E. miletus.


 
Lipophilicity/hydrophobicity of leptin protein in E. miletus
 
The leptin proteins of O. princeps inhabiting alpine tundra, T. belangeri from tropical regions, H. sapiens widely distributed across temperate zones and E. miletus were analyzed and compared (Fig 5). The four species share similar hydrophobic/hydrophilic fluctuation patterns in their leptin protein sequences. The leptin proteins of the North American pika and the large-eared vole exhibit stronger hydrophobicity, while those of humans and the T. chinensis display weaker hydrophobicity.

Fig 5: Prediction results of leptin affinity/hydrophobicity of O.


 
Comparison and analysis of leptin gene sequences in 56 mammalian species
 
phylogenetic analysis of leptin gene
 
The phylogenetic tree based on leptin gene sequences of 56 mammalian species (Fig 6) shows: Some Microtus species and E. miletus cluster into a small branch, with close genetic relationship and distant relationship with Dermoptera. Rodents of the same family cluster closely; squirrels, beavers and other rodents have longer branches and earlier differentiation than E. miletus. Ochotona and Lepus europaeus have short branches, adjacent to but independent of Rodentia. T. belangeri is close to Primates. Dermoptera species cluster into one branch, adjacent to Scandentia and Primates branches.

Fig 6: Phylogenetic tree of 56 mammalian leptin genes.


 
Distribution of 56 species in different temperature zones

The 56 species are clustered by their main distribution across temperature zones (Fig 7). Most primates, T. belangeri and Dermaptera cluster together, mainly in tropical areas. Two Ochotonidae pikas cluster, mainly in cold and sub-cold regions, differing from the two Leporidae species, which are mainly in temperate or temperate-subtropical zones. Most rodents are mainly in temperate zones; a few span tropical, subtropical and temperate zones, with some widely distributed from tropical to temperate.

Fig 7: Clustering heat map of the distribution of 56 species in each temperature zone.


 
Codon preference and GC content analysis
 
Analysis of ENC, GC3 and GC contents of leptin genes in 56 species (Table 1) showed: leptin gene GC content ranged 48.90-61.51 (average 54.76); GC3 was 64.07-81.55 (average 71.42), indicating a tendency to use G/C-ending codons (since the third codon rarely changes amino acid type); ENC values were 38.70-58.39 (average ~46.57). Lagomorpha had the highest GC and GC3 contents and the smallest ENC value, while E. miletus showed medium levels in all three.
 
Analysis of conservative areas and functional annotation areas
 
Multi sequence conservative motif analysis
 
The conservative motif prediction results of 56 leptin gene sequences were visualized with tbtools- aII (Fig 8). Motif 1-5 is stable in most species and its position is relatively concentrated. In rodents, motif 1-3 has more prominent repetition frequency and length; Some motifs are widely distributed in rodents and some primates and the distribution span is from 5'  to 3'. Some motifs only appear in specific groups and species, such as motif 7-11 unique to some Primates Macaca and are more densely distributed at the 3' end.

Fig 8: Results of conserved motif determination of 56 leptin gene sequences.



Functional annotation analysis of multiple sequence conservative motifs
 
Functional annotation was performed on motif 1-11, some conservative motifs had no functional annotation (Fig 9). Leptin was concentrated near the 20-25 amino acids. The region of membrane bound protein predicted to be outside the membrane, in the extracellular region, is expected to be located outside the membrane, occupying the posterior segment of the sequence (about 25-50 positions). Motif 1-3 showed the functional region of the conserved motif of the protein sequence encoded by the E. miletus leptin gene sequence.

Fig 9: Functional annotation results of each conserved motif.



Analysis of selection pressure and positive selection effect
 
The single ratio model selection pressure analysis (Table 3) shows that the overall ω=0.32993<1 and the leptin gene dN (1.5749) is significantly lower than dS (4.7733), indicating the presence of significant purification (negative) selection. E. miletus and Microtus gather together. In some branches, synonymous substitutions of rodents decrease, while in others, synonymous substitutions increase. In addition, the average content of GC3 between sequences is 0.7142.

Table 3: Reference values of leptin gene results under two branch models.


       
Free ratio model-based branch selection pressure analysis showed a better overall likelihood value (LNL = -5630.47) than the single ratio model (LNL = -5721.88). Likelihood ratio test (LRT) confirmed significant heterogeneity in selection pressure among branches. The overall kappa (ts/tv = 4.13) indicated a high transition/transversion ratio, consistent with mammalian gene evolution characteristics. With total nonsynonymous substitution rate (dN = 1.62) lower than synonymous substitution rate (dS = 4.55; dN < dS), the overall selection remained dominated by purifying selection. Under the free ratio model, Microtus and E. miletus (ω = 0.5661) clustered into one branch. Most species underwent strong purifying selection, while some branches showed significant positive selection.
 
Sequence characteristics of leptin gene and protein structure of E. Miletus adapted to their living needs
 
This study undertook an analysis of the leptin gene in E. miletus, predicting that its leptin protein contains a Sec/SPI-type signal peptide with a notably high support probability. Leptin functions by binding to its receptor (LepR), with α-helices playing a pivotal role in receptor recognition. The leptin of E. miletus exhibits a high α-helix ratio, which is conducive to a stable signal transduction domain. Research on Ochotona curzoniae has demonstrated that elevated α-helix ratios enhance receptor affinity and thermogenesis, facilitating adaptation to extreme cold conditions (Yang et al., 2011). Compared to humans and mice, E. miletus leptin displays a higher α-helix ratio, which is essential for energy regulation in adapting to high-altitude, cold environments. Its low random coil ratio indicates a compact structure, thereby reducing energy consumption. These findings, coupled with high sequence consistency with the leptin-LepR trimer template, confirm structural compatibility and enhance receptor binding efficiency in cold environments.
       
E. miletus leptin (127 amino acids) has the highest leucine proportion, potentially enhancing energy reserve and utilization in alpine environments (Zhang et al., 2009). Its amino acid composition is more ''energy-efficient'' than human leptin (≈11% leucine), aligning with survival needs in high-altitude, food-deficient habitats. As an unstable hydrophilic protein requiring continuous in vivo synthesis, its theoretical half-life indicates eukaryotic stability, ensuring sustained energy regulation signals. 
       
The hydrophobic region interacts with the phospholipid bilayer in transmembrane structures and maintains the conformational stability of membrane proteins at low temperatures (Tomczak et al., 2002). E. miletus exhibits high hydrophobicity, which facilitates protein structural maintenance and receptor signal transduction efficiency in cold environments (Chen et al., 2022). Plateau cold-zone species (O. princeps and E. miletus) showed strong hydrophobicity, ensuring stability and response speed of energy regulation signals. In contrast, temperate H. sapiens and tropical T. belangeri balance hydrophilicity and hydrophobicity, likely meeting the energy metabolism and environmental stability requirements.
 
Multidimensional characteristics of E. miletus adapted to living environment
 
Further evolutionary analysis showed that E. miletus and Microtus clustered, indicating leptin gene conservation in the same family and genus. Rodentia families have longer branches, with niche differentiation possibly driving leptin gene differentiation. Primates and hominids cluster closely, reflecting high leptin gene conservation.
       
In temperature distribution, Primates, T. belangeri and Dermoptera are mostly tropical. Ochotonidae and Leporidae differ in differentiation: some species range from tropical to temperate zones, with their leptin genes scattered in temperate clusters, showing environment-adaptive polymorphism to meet energy needs across temperature zones, reflecting molecular-level "ecological range expansion" (Londraville et al., 2014; Gong et al., 2013). E. miletus, mainly temperate, uses leptin gene regulation to adapt to the Hengduan Mountains’ extreme, climate-variable, large temperature-difference environment.
       
Molecular-level analyses of codon preference and conserved motifs further support these conclusions. The leptin gene shows a preference for G/C-ending codons; high GC3 content enhances DNA stability, aiding adaptation to UV-intense tropical environments and cold zones with large temperature fluctuations (Sambrook and Russell, 2001). A lower ENC value indicates stronger codon preference and faster translation (Sharp and Li, 1987); the moderate ENC value of E. miletus leptin suggests a trade-off between rapid translation and sequence variation, balancing core energy regulation functions with temperature zone specialization demands. Motifs 1-5, as core functional domains, ensure cross-species energy regulatory basics. The distribution of motifs 1-3 reveals E. miletus leptin retains signal peptide-guided secretion, metabolic regulation and extracellular membrane protein functions, linked to adaptation to the Qinghai-Tibet Plateau’s high-altitude, cold and hypoxic conditions (Chen et al., 2022; Gong et al., 2022). 
       
Selection pressure analyses showed overall purifying selection on the leptin gene, with highly conserved functions. Some Rodentia branches exhibited higher dS, indicating rapid evolution and sequence divergence, potentially linked to adaptive radiation. Most rodents clustered closely with short branch lengths and low genetic distances. Microtus clusters with E. miletus, supporting their close phylogenetic relationships. Leptin function is highly conserved among congeneric rodent species, with consistent patterns observed in other taxa.

CONCLUSION

This study elucidates the molecular mechanisms underlying Eothenomys miletus adaptation to high-altitude environments in the Hengduan Mountains. Key findings demonstrate that: Leptin gene evolution reflects long-term selective pressures under plateau conditions, with specific mutations stabilizing energy-regulation signaling pathways while maintaining metabolic flexibility to cope with resource fluctuations. Physiological specialization at the molecular level provides a paradigm for mammalian adaptation to extreme environments, bridging a critical gap between genetic changes and ecosystem-level resilience. These results offer two-fold implications: Theoretically, establishing leptin as a biomarker for high-altitude adaptation, challenging traditional views on metabolic adaptation timelines. Practically, proposing molecular targets for monitoring vulnerable plateau ecosystems, informing conservation strategies under climate change.

ACKNOWLEDGEMENT

This work was supported by the National Natural Scientific Foundation of China (No. 32160254), the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (NO. 202301 BA070001-076).
 
Ethical approval
 
All animal procedures were within the rules of Animals Care and Use Committee of School of Life Sciences, Yunnan Normal University. This study was approved by the committee (13-0901-011).

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Multidimensional Characterization and Adaptive Differentiation of Leptin Gene in Eothenomys miletus: Based on Bioinformatics and System Evolution Analysis

    H
    Huairan Wang1
    0009-0004-3687-4081
    W
    Wenrong Gao2
    https://orcid.org/0000-0001-9521-7613
    W
    Wanlong Zhu1,*
    0000-0001-8261-4089
    1School of Life Sciences, Yunnan Normal University, Kunming, 650500, China.
    2School of Biological Resources and Food Engineering, Qujing Normal University, Qujing, 655011, China.

    ABSTRACT

    Background: To investigate the differences between leptin gene in Eothenomys Miletus and other 55 mammal species, this study conducted a comparative analysis of multidimensional characteristics and adaptive differentiation of leptin genes.

    Methods: In this study, molecular biology experimental strategies have been used to extract and reverse transcription, gene cloning and sequencing of E. miletus and bioinformatics evaluation techniques have been used to analyze the sequence and structure, multi-sequence alignment and evolutionary evaluation of E. miletus and ecological and distribution statistical strategies had been used to analyze the leptin genes of 56 mammalian species such as E. miletus.

    Result: Leptin gene in E. miletus contained a Sec/SPI signal peptide, with α-helices dominating its structure. A model was constructed using leptin-LepR trimer (8 × 80.1. C leptin) as template. It encodes 127 amino acids with high proportions of leucine and serine; its molecular formula is C824H1353N221O252S8 (2658 atoms) with molecular weight of 18644.62 and theoretical isoelectric point of 5.86. The protein is unstable and hydrophilic, yet more hydrophobic than Tupaia chinensis and Homo sapiens. E. miletus was genetically close to Microtus, distant from Lagomorpha and clusters with temperate Rodentia. Its codon preference differed from Lagomorpha, with moderate GC content, GC3 content and ENC values. Motifs 1-5 were conserved in its leptin. Functional annotation shows regions related to signal peptide, leptin and extracellular domains. The ω value of ~0.5661 suggests evolutionary conservation. These results provide basis for exploring leptin gene evolution in E. miletus and mammals, offering perspective on mammalian adaptation to extreme environments and high-altitude protection.

    INTRODUCTION

    Leptin, a conserved protein hormone encoded by the obesity gene and primarily secreted by white adipose tissue (Priyadarshini et al., 2015), was identified in humans and mice in 1994 and has since become a key research focus. As a core regulator of energy metabolism, it modulates energy balance and suppresses food intake (Zhang et al., 2009; Sun, 2019), while also participating in immune responses (Wei et al., 2013), infectious diseases and reproduction (Jin et al., 2021; Zhang, 2015). Molecular and genetic studies have explored leptin genes in animals (Pang et al., 2016; Li et al., 2021; Meena et al., 2017), their associations with diseases (Yan et al., 2023; Ma, 2012) and leptin receptor genes (Yan et al., 2011; Li et al., 2011). Its physiological roles, linked to gene structural and functional variations, are critical for thermoregulation and metabolic heat production under environmental temperature stress. 
           
    Eothenomys miletus    , a Cricetidae rodent, which was endemic to the Hengduan Mountains of Yunnan (Han et al., 2021), it has been studied by our team for adaptive strategies, including physiological ecology, such as correlations between serum leptin levels and physiological parameters under varying conditions (Hou et al., 2020; Yang et al., 2013; Yang et al., 2016) and leptin-mediated regulation of body mass and energy metabolism via hypothalamic neuropeptides (Zhu, 2015). However, research on the molecular evolutionary mechanisms of its leptin gene remains limited. To clarify the leptin gene’s bioinformatics characteristics and evolutionary selection pressure, this study integrated leptin sequences from 55 mammals across five orders (Rodentia, Dermoptera, Lagomorpha, Scandentia, Primates) - taxa with wide distributions from equatorial tropics to cold zones, subject to strong temperature selection pressure. Recent studies note cold-dwelling Vulpes lagopus exhibits leptin receptor sensitivity, while tropical Tupaia belangeri has unique leptin expression regulation (Zhu et al., 2014), indicating temperature-driven adaptive evolution of leptin. However, the universality of such latitude- and temperature-related adaptations across taxa and cross-order evolutionary patterns of leptin genes, remain unclear. 
           
    This study analyzed the leptin gene’s protein structure and physicochemical properties in E. miletus and performed cross-order comparative analyses of leptin sequences from 56 mammals (including E. miletus) to reveal the gene’s molecular characteristics, adaptive evolutionary signals and cross-order evolutionary patterns in mammals.

    MATERIALS AND METHODS

    Obtaining the complete sequence of leptin gene from E. miletus
     
    From 2024 to June 2025, experiments and follow-up data analysis is conducted at the School of Life Sciences of Yunnan Normal University.
           
    Primers were designed according to the sequence of leptin gene in E. miletus. The upstream primer F was 5'- ATGTGCTGGGAGACTCCTGTG-3' and the downstream primer R was 3'- CAGCATCAGCTAAGGTCC-5'. Primers were synthesized by the company. The size of the amplified product was 503bp and the temperature of cleavage was 89.8oC. The steps and operations of extracting and purifying total RNA from hypothalamus in E. miletus was details in Zhu’s (2015). The whole sequence is transmitted to NCBI (GenBank No: pv838002).
     
    Sequence analysis of leptin gene in E. miletus
     
    Signal peptide prediction and protein secondary and tertiary structure analysis
     
    The sign peptide of leptin protein was once estimated by using SignalP-6.0 on-line tool, the secondary shape of leptin protein was once envisioned through SOPMA on line software and the tertiary shape of leptin protein used to be analyzed by means of SWISS-MODEL on line software.
     
    Analysis of physicochemical properties of leptin protein from E. miletus
     
    The hydrophilicity and hydrophobicity of leptin protein in E. miletus, Ochotona princeps, Tupaia belangeri and Homo sapiens were predicted and compared by using the ProtScale Program in the online software of ExPASY. The physical and chemical properties of rat leptin protein were analyzed by using the ProtParam program of ExPASY online software.
     
    Cross order comparison of leptin gene sequences in 56 species
     
    Collation sequence
     
    Leptin gene sequences from 55 mammalian species available on the NCBI platform, along with the newly obtained sequence from E. miletus, were organized (Table 1). These sequences are sorted out by means of techniques such as (Mao et al., 2022; Yang, 1997).

    Table 1: Basic information of 56 species of mammals and NCBI entry numbers.



    Construction of phylogenetic tree
     
    The systematic tree was constructed by the neighbor-joining (NJ) method and then the optimal model and the maximum likelihood method (ML) were used to construct the systematic tree and the self-expansion test (Bootstrap) was set to set 1000 iterations. Compare tree structures from different methods to ensure accuracy. Beautify the phylogenetic tree using Chiplot online software.
     
    Distribution analysis of 56 species in five temperature zones
     
    From the perspective of ecological and biological distribution, the main distribution of species is stable survival and reproduction, with large population, high density and wide distribution range. If the species are mainly distributed in a certain temperature zone, it is recorded as 1 and other distributions are recorded as 0. With the support of animal geographical distribution, ecological habits, professional literature and database, the main distribution of 56 species in 5 temperature zones were counted by Excel. Chiplot was used to analyze the clustering of 56 species in each temperature zone.
     
    Codon preference and determination of GC content
     
    The number of effective codons (ENC), GC content and third GC content (GC3s) of the sequence were calculated using the online tool EMBOSS Explorer.
     
    Conservative region determination and functional annotation analysis of multiple sequences
     
    MEME online software is used to predict the conservative motifs of multiple sequences, compare the predicted conservative motifs with Pfam, PROSITE and other databases, annotate the functional domains corresponding to the motifs and visualize the conservative regions through TBtools - II.
     
    Select pressure detection
     
    PAML software was used to calculate the ratio of nonsynonymous replacement rate (dN) and synonymous replacement rate (dS) (ω=dN/dS) to measure the selection pressure of leptin gene in the process of evolution. The single ratio (M0) and free ratio (M1) branching models that rely on likelihood ratio test (LRT) in Codeml program are used to detect the overall selection pressure of leptin gene and whether there is heterogeneity of selection pressure in different evolutionary branches of leptin gene.

    RESULTS AND DISCUSSION

    Bioinformatics analysis of leptin in E. miletus
     
    Signal peptide assay
     
    It showed that there was a highly reliable Sec/SPI type signal peptide in the leptin protein of E. miletus (Fig 1). The probability distribution of different regions of signal peptide is high and the probability of non-signal peptide region is low, which is consistent with the judgment of signal peptide. Cleavage site: It is predicted to be between the 21st and 22nd amino acids, with a probability of up to 0.978790. It has high reliability and the cleavage site is clear, which supports the functional localization of its secreted protein. Signal peptide type: it belongs to Sec/SPI signal peptide and the probability of signal peptide is 0.9998.

    Fig 1: Prediction results of leptin signal peptide in the E. miletus.


     
    Protein secondary and tertiary structure prediction
     
    protein secondary structure prediction results
     
    After removal of the signal peptide sequence, the mature leptin protein of E. miletus consists of 146 amino acid residues. Secondary structure prediction using SOPMA (Fig 2) revealed a predominance of α-helices (h), comprising 94 residues and accounting for 64.38% of the structure (Fig 3). Random coils (c) were the secondary major structural element, with 50 residues, while the proportion of extended strands (e) was minimal at only 1.37%.

    Fig 2: The distribution of α-spiral (blue vertical line), random curl (orange short bar) and extended strand (purple vertical line) in the sequence.



    Fig 3: Prediction results of secondary structure of leptin protein in E. miletus.


     
    Prediction results of protein tertiary structure
     
    SWISS-MODEL was used to construct the tertiary structure model of leptin protein in E. miletus (Fig 4). The core template: leptin LEPR trimer structure (8  × 80.1. C leptin) was selected to establish the model. The sequence consistency was as high as 82.04%, the global model quality score (GMQE) was 0.76 and qmeandico global was 0.76±0.07, indicating that the quality of the model was good and the construction was reasonable. The spiral structure is prominent in the modeling and the model presents a spatial folding shape dominated by α - helix, with less random curl.

    Fig 4: Three-dimensional structure prediction result of leptin protein in E. miletus.


     
    Determination and analysis of physical and chemical properties
     
    The analysis results of physical and chemical properties of rat leptin protein showed that the number of amino acids was 127, including 20 essential amino acids, of which leucine accounted for the highest proportion and serine accounted for the second highest proportion (Table 2). Its molecular formula is C824H1353N221O252S8, with a total atomic number of 2658 and a molecular weight of 18644.62. The theoretical isoelectric point of protein is 5.86, which is acidic. The theoretical half-life of E. miletus leptin protein in vitro (mammalian reticulocytes) was 30 h, in yeast > 20 h and in Escherichia coli > 10 h. The aliphatic index of the protein was 119.04; Average hydrophilicity (GRAVY): 0.113, instability index: 50.89, which is an unstable hydrophilic protein.

    Table 2: Amino acid composition of leptin protein in E. miletus.


     
    Lipophilicity/hydrophobicity of leptin protein in E. miletus
     
    The leptin proteins of O. princeps inhabiting alpine tundra, T. belangeri from tropical regions, H. sapiens widely distributed across temperate zones and E. miletus were analyzed and compared (Fig 5). The four species share similar hydrophobic/hydrophilic fluctuation patterns in their leptin protein sequences. The leptin proteins of the North American pika and the large-eared vole exhibit stronger hydrophobicity, while those of humans and the T. chinensis display weaker hydrophobicity.

    Fig 5: Prediction results of leptin affinity/hydrophobicity of O.


     
    Comparison and analysis of leptin gene sequences in 56 mammalian species
     
    phylogenetic analysis of leptin gene
     
    The phylogenetic tree based on leptin gene sequences of 56 mammalian species (Fig 6) shows: Some Microtus species and E. miletus cluster into a small branch, with close genetic relationship and distant relationship with Dermoptera. Rodents of the same family cluster closely; squirrels, beavers and other rodents have longer branches and earlier differentiation than E. miletus. Ochotona and Lepus europaeus have short branches, adjacent to but independent of Rodentia. T. belangeri is close to Primates. Dermoptera species cluster into one branch, adjacent to Scandentia and Primates branches.

    Fig 6: Phylogenetic tree of 56 mammalian leptin genes.


     
    Distribution of 56 species in different temperature zones

    The 56 species are clustered by their main distribution across temperature zones (Fig 7). Most primates, T. belangeri and Dermaptera cluster together, mainly in tropical areas. Two Ochotonidae pikas cluster, mainly in cold and sub-cold regions, differing from the two Leporidae species, which are mainly in temperate or temperate-subtropical zones. Most rodents are mainly in temperate zones; a few span tropical, subtropical and temperate zones, with some widely distributed from tropical to temperate.

    Fig 7: Clustering heat map of the distribution of 56 species in each temperature zone.


     
    Codon preference and GC content analysis
     
    Analysis of ENC, GC3 and GC contents of leptin genes in 56 species (Table 1) showed: leptin gene GC content ranged 48.90-61.51 (average 54.76); GC3 was 64.07-81.55 (average 71.42), indicating a tendency to use G/C-ending codons (since the third codon rarely changes amino acid type); ENC values were 38.70-58.39 (average ~46.57). Lagomorpha had the highest GC and GC3 contents and the smallest ENC value, while E. miletus showed medium levels in all three.
     
    Analysis of conservative areas and functional annotation areas
     
    Multi sequence conservative motif analysis
     
    The conservative motif prediction results of 56 leptin gene sequences were visualized with tbtools- aII (Fig 8). Motif 1-5 is stable in most species and its position is relatively concentrated. In rodents, motif 1-3 has more prominent repetition frequency and length; Some motifs are widely distributed in rodents and some primates and the distribution span is from 5'  to 3'. Some motifs only appear in specific groups and species, such as motif 7-11 unique to some Primates Macaca and are more densely distributed at the 3' end.

    Fig 8: Results of conserved motif determination of 56 leptin gene sequences.



    Functional annotation analysis of multiple sequence conservative motifs
     
    Functional annotation was performed on motif 1-11, some conservative motifs had no functional annotation (Fig 9). Leptin was concentrated near the 20-25 amino acids. The region of membrane bound protein predicted to be outside the membrane, in the extracellular region, is expected to be located outside the membrane, occupying the posterior segment of the sequence (about 25-50 positions). Motif 1-3 showed the functional region of the conserved motif of the protein sequence encoded by the E. miletus leptin gene sequence.

    Fig 9: Functional annotation results of each conserved motif.



    Analysis of selection pressure and positive selection effect
     
    The single ratio model selection pressure analysis (Table 3) shows that the overall ω=0.32993<1 and the leptin gene dN (1.5749) is significantly lower than dS (4.7733), indicating the presence of significant purification (negative) selection. E. miletus and Microtus gather together. In some branches, synonymous substitutions of rodents decrease, while in others, synonymous substitutions increase. In addition, the average content of GC3 between sequences is 0.7142.

    Table 3: Reference values of leptin gene results under two branch models.


           
    Free ratio model-based branch selection pressure analysis showed a better overall likelihood value (LNL = -5630.47) than the single ratio model (LNL = -5721.88). Likelihood ratio test (LRT) confirmed significant heterogeneity in selection pressure among branches. The overall kappa (ts/tv = 4.13) indicated a high transition/transversion ratio, consistent with mammalian gene evolution characteristics. With total nonsynonymous substitution rate (dN = 1.62) lower than synonymous substitution rate (dS = 4.55; dN < dS), the overall selection remained dominated by purifying selection. Under the free ratio model, Microtus and E. miletus (ω = 0.5661) clustered into one branch. Most species underwent strong purifying selection, while some branches showed significant positive selection.
     
    Sequence characteristics of leptin gene and protein structure of E. Miletus adapted to their living needs
     
    This study undertook an analysis of the leptin gene in E. miletus, predicting that its leptin protein contains a Sec/SPI-type signal peptide with a notably high support probability. Leptin functions by binding to its receptor (LepR), with α-helices playing a pivotal role in receptor recognition. The leptin of E. miletus exhibits a high α-helix ratio, which is conducive to a stable signal transduction domain. Research on Ochotona curzoniae has demonstrated that elevated α-helix ratios enhance receptor affinity and thermogenesis, facilitating adaptation to extreme cold conditions (Yang et al., 2011). Compared to humans and mice, E. miletus leptin displays a higher α-helix ratio, which is essential for energy regulation in adapting to high-altitude, cold environments. Its low random coil ratio indicates a compact structure, thereby reducing energy consumption. These findings, coupled with high sequence consistency with the leptin-LepR trimer template, confirm structural compatibility and enhance receptor binding efficiency in cold environments.
           
    E. miletus     leptin (127 amino acids) has the highest leucine proportion, potentially enhancing energy reserve and utilization in alpine environments (Zhang et al., 2009). Its amino acid composition is more ''energy-efficient'' than human leptin (≈11% leucine), aligning with survival needs in high-altitude, food-deficient habitats. As an unstable hydrophilic protein requiring continuous in vivo synthesis, its theoretical half-life indicates eukaryotic stability, ensuring sustained energy regulation signals. 
           
    The hydrophobic region interacts with the phospholipid bilayer in transmembrane structures and maintains the conformational stability of membrane proteins at low temperatures (Tomczak et al., 2002). E. miletus exhibits high hydrophobicity, which facilitates protein structural maintenance and receptor signal transduction efficiency in cold environments (Chen et al., 2022). Plateau cold-zone species (O. princeps and E. miletus) showed strong hydrophobicity, ensuring stability and response speed of energy regulation signals. In contrast, temperate H. sapiens and tropical T. belangeri balance hydrophilicity and hydrophobicity, likely meeting the energy metabolism and environmental stability requirements.
     
    Multidimensional characteristics of E. miletus adapted to living environment
     
    Further evolutionary analysis showed that E. miletus and Microtus clustered, indicating leptin gene conservation in the same family and genus. Rodentia families have longer branches, with niche differentiation possibly driving leptin gene differentiation. Primates and hominids cluster closely, reflecting high leptin gene conservation.
           
    In temperature distribution, Primates, T. belangeri and Dermoptera are mostly tropical. Ochotonidae and Leporidae differ in differentiation: some species range from tropical to temperate zones, with their leptin genes scattered in temperate clusters, showing environment-adaptive polymorphism to meet energy needs across temperature zones, reflecting molecular-level "ecological range expansion" (Londraville et al., 2014; Gong et al., 2013). E. miletus, mainly temperate, uses leptin gene regulation to adapt to the Hengduan Mountains’ extreme, climate-variable, large temperature-difference environment.
           
    Molecular-level analyses of codon preference and conserved motifs further support these conclusions. The leptin gene shows a preference for G/C-ending codons; high GC3 content enhances DNA stability, aiding adaptation to UV-intense tropical environments and cold zones with large temperature fluctuations (Sambrook and Russell, 2001). A lower ENC value indicates stronger codon preference and faster translation (Sharp and Li, 1987); the moderate ENC value of E. miletus leptin suggests a trade-off between rapid translation and sequence variation, balancing core energy regulation functions with temperature zone specialization demands. Motifs 1-5, as core functional domains, ensure cross-species energy regulatory basics. The distribution of motifs 1-3 reveals E. miletus leptin retains signal peptide-guided secretion, metabolic regulation and extracellular membrane protein functions, linked to adaptation to the Qinghai-Tibet Plateau’s high-altitude, cold and hypoxic conditions (Chen et al., 2022; Gong et al., 2022). 
           
    Selection pressure analyses showed overall purifying selection on the leptin gene, with highly conserved functions. Some Rodentia branches exhibited higher dS, indicating rapid evolution and sequence divergence, potentially linked to adaptive radiation. Most rodents clustered closely with short branch lengths and low genetic distances. Microtus clusters with E. miletus, supporting their close phylogenetic relationships. Leptin function is highly conserved among congeneric rodent species, with consistent patterns observed in other taxa.

    CONCLUSION

    This study elucidates the molecular mechanisms underlying Eothenomys miletus adaptation to high-altitude environments in the Hengduan Mountains. Key findings demonstrate that: Leptin gene evolution reflects long-term selective pressures under plateau conditions, with specific mutations stabilizing energy-regulation signaling pathways while maintaining metabolic flexibility to cope with resource fluctuations. Physiological specialization at the molecular level provides a paradigm for mammalian adaptation to extreme environments, bridging a critical gap between genetic changes and ecosystem-level resilience. These results offer two-fold implications: Theoretically, establishing leptin as a biomarker for high-altitude adaptation, challenging traditional views on metabolic adaptation timelines. Practically, proposing molecular targets for monitoring vulnerable plateau ecosystems, informing conservation strategies under climate change.

    ACKNOWLEDGEMENT

    This work was supported by the National Natural Scientific Foundation of China (No. 32160254), the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (NO. 202301 BA070001-076).
     
    Ethical approval
     
    All animal procedures were within the rules of Animals Care and Use Committee of School of Life Sciences, Yunnan Normal University. This study was approved by the committee (13-0901-011).

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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