Assignment 4

Analysis of recent paper relating to leptin

Paper:

Yasui, T., Tanaka, N., Shimizu, F., Minakuchi, M., Matsuzaki, T., Kuwahara, A., Iwasa, T., Irahara, M., Furumoto, H.(2008). Transition of Leptin receptor expression during pubertal development in female rat pituitary. Endocrine Journal, 55(1); 191-198.

Link to paper

It is known that the hormone leptin plays an important role in sexual maturation and thus reproduction. This has been shown in mice with mutant ob alleles that did not sexually mature and were infertile. After leptin treatment the mice became fertile. Leptin's role in sexual maturation was also seen in underweight females that experienced delayed puberty. In females with adequate body fat at the onset of puberty the concentration of serum leptin was found to increase before that of other hormones neccessary for puberty. This suggests that a certain mass of adipose tissue or fat stores are required for proper sexual maturation and since the source of leptin is from adipocytes, leptin secretion must have some role in puberty.

Leptin's involvement in sexual maturation and reproduction is through leptin receptors in the hypothalamic-pituitary neurons. In a prior study by Yasui and collegues, they determined that leptin stimulated LH and FSH production and secretion from rat pituitary cells. In their study that will be discussed, Yasui et al. were focused on the expression of leptin receptor (ob-Rb) in female rat pituitary. The ob-Rb is the active form of leptin receptor in that its able to cause signal transduction while the inactive leptin receptor is the ob-Ra form. The active form (ob-Rb) is found on LH, FSH, TSH, and GH secreting cells of the pituitary. In this study the objective was to determine the expression level of the ob-Ra and ob-Rb receptors in female rat pituitary from the juvenile and puberty, to the mature stages[1].

>Their study involved three seperate experiments, of which the first used female rats of 4, 6 and 8 weeks of age (juvenile, pubertal and mature stage). The LH, FSH, estradiol and leptin concentrations in serum samples from the rats were first determined and both pituitary and cerebrum tissue were obtained for real time quantitative PCR analysis for mRNA levels of ob-Ra and ob-Rb.

>In the second experiment, pituitaries were also obtained from 4 week old(juvenile) female rats to determine relative locations of leptin receptor and LH by use ot double labeling immunohistochemistry (antibody labeling).

>The last experiment was done to determine the effects of various hormones that relate to pubertal development on ob-Rb expression in pituitary cells of juvenile (4 week old) rats. The pituitary cells were incubated with the following hormones: leptin, GnRH, insulin, estradiol, GnRH antagonist, Inhibin A, GHRH, and nicotine (stimulate ob-Rb expression in rat hypothalamus).

Results summary
1. The results for measurments of baseline leptin, FSH, LH and estradiol concentrations revealed that the concentration of theses hormones were much higher in the 6 and 8 week old rats than the 4 week olds. The serum LH concentration for the three different ages were too low for detection and thus were not recorded. The concentration of each hormone increased the most from the juvenile to pubertal stage, confirming leptin's action in stimulation of gonadotropin release at the onset of puberty.

Table taken from Yasui T., et al. 2008 Transition of leptin receptor expression during pubertal development in female rat pituitary . Endocrine Journal 55(1); 191-198

2. The quantitative real time PCR analysis results showed that expression levels of ob-Ra (inactive) and ob-Rb (active) receptors in the cerebrum were not different. As well, there was no significant different in expression levels between the juvenile, pubertal and mature ages for each receptor. There was also no significant difference in expression levels of ob-Ra receptor between the three different stages in the rat pituitary. However, there was a significant difference in ob-Rb(active) receptor expression levels in the pituitary between the juvenile and mature stages. Figure 1 B of the paper shows that ob-Rb expression in rat pituitary decreased from the juvenile to mature stage.

Figure showing the mRNA expression of ob-Ra and ob-Rb in female rat pituitary at juvenile, pubertal and mature stages of development from the results (Figure 1) of Yasui, T., 2008. Transition of leptin receptor expression during pubertal development in female rat pituitary. Endocrine Journal 55(1); 191-198

3. The results of the study also confirmed that rat pituitary cells with the ob-Rb receptor also produced LH, shown by the immunofluorescence overlay of the double antibody labelling results.

4. Finally, the results showed that the expression level of ob-Rb in the presence of each hormonal factor tested did not differ significantly from the control level in the juvenile stage before puberty and thus none of these factors were found to contribute to the high level of ob-Rb receptors in juvenile rat pituitary.

Conclusions

The expression level of ob-Rb in the cerebrum remained constant from juvenile to the mature stage. This can indicate that ob-Rb receptor expression must have increased before the juvenile stage in hypothalamic neurons. Thus leptin-stimulated GnRH secretion is likely not regulated by changes in hypothalamic ob-Rb expression in puberty, but instead that it could be regulated by leptin concentration.

Since ob-Rb expression levels in the pituitary were highest in the juvenile stage when the leptin concentration was lowest, high ob-Rb expression did not correlate with high amounts of its ligand leptin. The physiological significance of high ob-Rb expression at this stage (juvenile) was likely to accomodate the surge in serum leptin concentration later, at the onset of puberty. It was also concluded that leptin receptor in the rat pituitary could be regulated by the different developmental stages. This is very likely as the expression level of different genes vary at different stages of development and expression level of other genes may affect that of ob-Rb at different stages of development. The results of the third experiment allowed the authors to conclude that none of the factors tested actually had an effect on the ob-Rb expression at the juvenile stage.

Brief Critique

- All tables and figures in the results of the paper were presented clearly, although the differences in units of concentration for the hormones in Table 1 were inconvenient when comparing the hormone concentrations.

- The disscusion of results in the paper was very brief and did not really outline how the results are applicable to future experiments in this area of research. Also, the results for experiment 3 (effect of various factors on expression of ob-Rb mRNA in cultured pituitary cells) did not anwser the posed question of what could influence the ob-Rb expression in the juvenile rat pituitary cells. The authors explained these results as having been affected by experimental errors such as inadequate culture time or "other factors" which may be involved in ob-Rb expression.

- Overall the results and methods of the study were presented well and full details of the techniques used in the experiments such as RT-PCR and the immunohistochemical double labeling were explained . The authors also explained the necessary background infromation about the leptin receptors ob-Ra/ob-Rb. As well, they referred to the results of past experiments, and clearly explained the objectives of their study.

Future experiment

It may be possible that ob-Rb receptors are expressed in female rat ovary. This would mean that leptin could then affect estradiol production though these receptors, in addition to those in the hypothalamus and pituitary. This could be determined by analysis of female rat ovarian tissue by real time quantitative PCR or northern blotting for the leptin receptor gene. If the results show expression of leptin receptors in ovary, then the expression of these receptors may also be regulated developmentally.

If leptin, FSH, estradiol concentrations and leptin receptor expression shows the same pattern in humans, it may help explain the mechanism of puberty onset. To determine this, a long term study would need to be performed similar to that described above.

Assignment 3
Function/Pathology

The main function of Leptin in the body is to maintain energy balance and thus body weight by controlling the appetite (amount of food intake) and stimulating the break down of energy. Leptin does so by interaction with receptors (LR) in the hypothalamus. There are two specific groups of neurons in the hypothalamus that have receptors for the leptin hormone. These include the agouti-related peptide (AgRP), neuropeptide Y(NPY) producing neurons and a group of neurons that produce pro-opiomelanocortin (POMC). AgRP and NPY stimulate the appetite as they are orexigenic, while POMC acts to decrease appetite (anorexigenic)[2].





Appetite
Leptin hormone in circulation easily crosses the blood-brain barrier and binds to its receptors on the two groups of neurons described. This results in activation (depolarization) of POMC producing neurons which increase the synthesis of POMC and melanocortins[2]. In contrast, leptin binding to receptors on AgRp and NPY neurons has an inhibitory effect and decreases the production of AgRp and NPY[2]. The overall effect is decreased appetite and an increase in energy metabolism. In the case of decreased leptin levels, the appetite is stimulated because of less inhibition of the appetite stimulators (AgRp and NPY) and decreased stimulation of POMC synthesis. In addition to the two main groups of neurons mentioned, leptin receptors have also been found on other neurons that produce neuropeptides which have an effect on food intake[4]. These other neuropeptides are all anorexigenic and are listed in the table below(effect of leptin on neuropeptide expression) along with NPY and POMC/melanocortins.

Energy Metabolism

Along with suppressing the appetite, leptin can alter energy metabolism in order to maintain an energy balance. Like some of the cytokines, leptin can act to stimulate the sympathetic nervous system (SNS). The stimulation of SNS causes release of epinephrine which increases the breakdown of fat into free fatty acids(lipolysis) and inhibits division of preadipocytes[3]. Insulin levels are also reduced by epinephrine, an effect also seen in response to increased leptin. This indicates an indirect mechanism of energy balance by leptin through the SNS. The regulation of energy metabolism is also attributed to leptin's effect on the thyrotropin-releasing hormone (TRH) levels. High leptin levels increase TRH levels and thus the level of thyroid stimulating hormone (TSH)[2]. This triggers production of thyroxine and thus increased metabolic rate (energy metabolism).

Reproduction
Leptin is found to be an important hormone in regulation of the reproductive system. This is because leptin can signal to the brain whether or not fat (energy) stores are high enough for reproduction. Leptin does this through the gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus which signals releas of FSH and LH from the pituitary[2]. Leptin's role in regulation of reproductive hormones has been shown in mice with two mutant alleles for the leptin(ob) gene. These mutant mice had very low fertility, but injecting recombinant leptin hormone increased the ability of these mice to reproduce[4].

Immune system

The molecular properties of leptin are very similar to the cytokines which are messenger proteins used by the immune system. Leptin has a direct effect on the immune system by increasing production of monocytes (develop into macrophages) and the release of cytokines that induce the inflammatory immune response (IL-6 and TNFalpha)[3]. As well, most stem cells that give rise to blood cells (hematopoietic cells) have the leptin receptor. Leptin-receptor binding on these cells stimulates production of blood cells and thus cells of the immune system (white blood cells)[3].

Pathology

Obesity
Abnormal levels of leptin hormone or its receptor (LR) can result in high energy intake and low energy metabolism which can equate to loss of the energy (body fat) balance mechanism. High amounts of body fat leading to obesity can be caused by mutaion in the leptin gene (ob) or the leptin receptor gene(db). In addition to excess body fat, supression of the immune response, reduced sexual maturity, insulin resistance and reduced thermoregulation are also observed in animals with a mutation in the leptin or leptin receptor gene[1]. Obesity due to mutations in the leptin or leptin receptor gene can be detected by measuring the levels of leptin in the blood. If the result is abnormally high/increased leptin levels the obese state is likely caused by mutations in the leptin receptor which causes a resistance to leptin. Very low leptin levels in comparison to normal levels for the weight of the individual indicates that are mutations in the leptin (ob) gene which leads to low levels of circulating leptin[3].

The obese state caused by defects in the leptin hormone or leptin receptor, can manifest other conditions such as diabetes, cardiovascular disease or reduced sexual maturation[1]. Given this, measurement of blood leptin may be ueseful for diagnosing reproductive disorders or assesing the risk level for certain cardiovascular diseases.

References

1. Ahima, R.,S. (2002). Obesity gene therapy: slimming immature rats. Gene Therapy. 10:196-197. Retrieved Nov. 3, 2008 from http://www.nature.com/gt/journal/v10/n3/full/3301920a.html

2. Robertson, D., Leinninger, G., Myers, M.(2008). Molecular and neural mediators of leptin action. Physiology & Behavior. 94: 637-642

3. Robinson, C., Kordon, D., & Hanoune, J. (2002). Brain Somatic Cross-Talk and the Central Control of Metabolism. Germany. Springer-Verlag, Berlin, Heidelberg.

4. Trayhurn, P., Mercer, J., & Rayner, D. (1999, February). Leptin: Fundamental aspects. International Journal of Obesity & Related Metabolic disorders, 23, s22.

Assignment 2

Structure

The Leptin protein is encoded by the ob gene on chromosome 7 (127.67-127.68 Mb) of humans. The gene encodes a polypeptide with 167 amino acids and a weight of 16 kDa [2]. The 167 amino acid sequence has a 21 amino acid signal sequence which is cleaved in post translational processing and a 147 amino acid protein circulates in the blood [1].

The secondary structure of the circulating protein is an alpha helix in which the amino acid residues interact by hydrogen bonds. The tertiary structure consists of a helical bundle that consists of four alpha helical domains. These domains are held together by several loop structures that all differ in length [2]. These loop structures allow two of the alpha helices to be antiparallel meaning that their sequences of amino acids run in opposite directions (N-terminals on opposite ends). The other domains run in the same direction so that overall the protein has the first two helical domains pointing upward while the other two domains are pointing downward as visible in the Figure above.


The core of the tertiary structure (helix bundle) contains a region of hydrophobic residues
(Val, Ile, Met, Ala) within two of the helices. These residues interact with leu,val residues contained in the longest loop structure as well as with each other [2]. The hydrophobic interactions between the residues and their position at the top of the bundle contribute to stabilization of the protein core and thus the protein as a whole.

Leptin's structure is most similar to that of the cytokines, particularly Interleukin-6 (Il-6) which can induce or inhibit inflammatory immune responses. The granulocyte colony stimulating factor (G-CSF) is also very similar to leptin in terms of molecular structure. G-CSF is also a cytokine/growth factor that has immune function in the development of bone marrow.

Just as leptin is simlar to the cytokines, its receptor is similar to the cytokine receptors and it is classified under the cytokine receptor family (Class I). These receptors have an immunoglobulin like domain in the middle of two CRH domains as well as two fibronectin domains (type III) [3]. The mechanism of action for the receptors is by Janus Kinase 2 (Jak2)/STAT pathway where hormone binding causes recruitment of Jak2 and phosphorylation of receptro tyrosines. This recruits the transcription factor STAT which is also phoshorylated (activated), binds to certain promoters and activates transcription of specific genes.

Leptin-Leptin receptor interactions
Leptin-receptor interactions involve three binding sites located at different helices in the protein, which was shown by Zabeau et al in an experiment that involved the mapping of leptin- receptor binding sites. The helices of the protein are named as helix A through D and two of the binding were found to be within the helix D. A third binding site involves two helices, A and C which associates with one of the CRH domains on the receptor. The binding sequences located at the end of the D helix associate with the immunoglobulin like region of receptor [3]. A model of leptin- leptin receptor interactions is shown in the figure below.

Leptin interaction with receptor (LR)

Model of Class I cytokine receptor

Leptin protein alignment

The degree of correlation of leptin hormone structure between different closely related species can be important in explaining the various effects of leptin which can differ between species. It is interesting to see how close in molecular structure, a hormone can be (in this case leptin), between different species.

Protein alignments for leptin were obtained using Blast and ClustalW from which alignment scores were obtained between each species selected. A high score indicates a high degree of similarity between sequences. Figure 1 shows the actual protein sequence alignment from which the location of identical residues between the sequences can be seen. The alignment scores in Table 1 shows that leptin hormone is highly conserved among mammals. Overall the alignment scores correlate with the phylogenetic data shown in Figure 2. The human hormone shows a high similarity to the mouse with 82% score while it has the lowest similarity with the rabbit (77%). Conservation of the hormone sequence is also shown by the high alignment score between human and the cat which is on a different evolutionary lineage as shown in Figure 2.

Figure 1: Sequence alignment of leptin hormone in different mammal species produced using ClustalW

Figure 2: Phylogenetic Tree

References

1 Ruffing, R., & Bardot, J. 2007. Leptin E-100, the obese protein. Retrieved October 18, 2008 from the world wide web: http://biology.kenyon.edu/BMB/Chime2/2005/Leptin%20Model/Frames/start.html

2 Wikipedia, 2008. Leptin. Retrieved October 18, 2008 from the world wide web: http://en.wikipedia.org/wiki/Leptin

3 Zabeau, L., Vandekerckhove, J., Tavernier, j., Peelman, F., Iserentant, H., and De Smet A.S. 2006. Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin -leptin receptor complex. Journal of Biol. Chem. Jun 2:28/(22): 15496-504

Assignment 1

My favourite hormone

Leptin is a peptide hormone produced in the adipocytes of adipose tissue. It is also known to be produced in the stomach, placenta and some fetal tissues [3]. The polypeptide is encoded by the ob gene and the resulting protein has four alpha helical domains which makes it similar to the class-1 family of cytokines [1].

The main function of leptin is to regulate adipose tissue or fat mass by suppression of the appetite and increase of energy metabolism. Leptin also plays a part in reproduction and the immune system. The actions of leptin are acheived by communication with neurons in the hypothalamus where leptin receptors (LepR) are located. The amount of leptin in cirulation is proportional to the amount of fat stores in the body [2]. By interacion with neurons associated with other hormones, leptin works with the hypothalamus to govern the balance of energy intake and energy metabolism according to how much energy stores are already present.

www.nurseminerva.co.uk/images/leptin2.jpg

The Leptin Feedback Loop

Leptin hormone was first observed and its DNA encoding sequence determined in 1944 by Dr. J Friedman(Rockefeller University) and others. They cloned a mutant form of the ob gene from an obese mouse. The mice with mutations in both ob genes became very fat and inactive. They administered what is now called leptin and the result was weight loss and decreased food consumption in the mice [3]. This experiment provided evidence that body weight must be under hormonal control and raised the question of whether or not leptin plays a role in other regulatory mechanisms.

References

1. Robertson, S., Myers, M., Leinninger, G. (2008). Molecular and neural mediators of leptin action. Physiology & Behavior. 94: 637-642.

2. Robinson, C., Kordon, D., & Hanoune, J. (2002). Brain Somatic Cross-Talk and the Central Control of Metabolism. Germany. Springer-Verlag, Berlin, Heidelberg. pp.15-17

3. Trayhurn, P., Mercer, J.,& Rayner, D. (1999, February). Leptin: Fundamental aspects. International Journal of Obesity & Related Metabolic Disorders, 23, s22.