Cloning and expression analysis of leptin and its receptor in the axolotl (Ambystoma mexicanum)

Abstract

Since its discovery in 1994, the adipose tissue hormone leptin has been well established as a key regulator of energy balance in mammals. However, little is known about the molecular evolution of the hormone and its function in non-mammalian vertebrates. This project builds on the recent identification of leptin in an amphibian, the tiger salamander, to investigate the leptin signalling system in a laboratory salamander, the axolotl. The overall aim of the project was to obtain cDNA sequences of the axolotl leptin and leptin receptor (LEPR) genes, to analyse their expression and to study their expression due to nutritional state. Cloning the axolotl LEPR was a key component of the work because no sequence information was previously available. Semi-degenerate primers were used to clone a 248 bp fragment of the LEPR, which shared 62% identity with human leptin at the amino acid level. Attempts to obtain the full-length cDNA sequence were unsuccessful. However, the sequence grouped in proximity to a Xenopus LEPR in a phylogenetic tree, and Northern hybridization revealed a transcript size of approximately 3 kb, which corresponded with that of other vertebrate LEPRs. To establish the expression pattern of leptin and the LEPR between tissues, quantitative real-time PCR was performed in two different age groups of animals. In adults, the highest expression of leptin was observed in the fat, brain and heart whereas in juveniles leptin expression was significantly higher in the fat body compared to all other tissues. The highest expression of LEPR was found in the brain and skeletal muscle. These findings agree with the main sites of leptin and LEPR expression in mammals, Xenopus, and fish providing further evidence that the gene fragments cloned represents the axolotl leptin and LEPR. In order to understand the possible role(s) of leptin in the regulation of food intake and energy metabolism in amphibians, changes in leptin and LEPR expression due to nutritional state were investigated. Short-term fasting did not result in any significant changes in leptin expression in the fasted animals, nevertheless it showed a tendency towards a lower leptin and LEPR expression of fasted axolotls. These findings indicate that the regulation of leptin expression by nutritional state more closely resemble the situation in other ectotherms such as teleost fish. This work provides the opportunity to explore how the physiological functions of leptin have changed during evolutionary history.