Review Paper on : Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feeding Behavior
Past research has shown that the hypothalamus could play a critical role in feeding and energy homeostasis. This paper attempts to specify the role of the hypothalamus by studying a set of G protein-coupled receptors (GPCRs) and their binding peptides. It had been found that the family of seven transmembrane GPCRs responded to a wide variety of molecules, but the specific binding molecules of many GPCRs are not known. These ÒorphanÓ GPCRs were thought to bind to some small regulatory peptides, but many of these potential peptides remained undescribed. To try to determine the binding and activation of these orphan GPCRs, a cell based reporter system was used to screen for these endogenous peptide ligands and their specific orphan GPCRs. This research team located two novel neuropeptides (orexin-A and -B) that bind and activate two closely related (previously) orphan GPCRs. After characterizing these neuropeptides and their receptors, tests were run that attempted to connect these peptides and receptors to the brainÕs control of feeding and energy homeostasis. The following is a description of the researchers methodology and results.
The first step taken by the researchers was to screen for peptide/receptor binding and activity. Figure 1 shows the results of the screening and subsequent purification of the endogenous ligands for the (previously) orphan GPCR HFGAN72 (OX,R). To test for peptide/receptor binding and activity, high-resolution HPLC fractions of various tissue extracts were screened for GPCR agonist activity using multiple orphan GPCR-expressing cell lines. Over fifty transfectant cell lines (each containing cDNA from distinct orphan GPCRs) were challenged with HPLC fractions derived from various tissues, and calcium activity was monitored. Figure 1 A (bottom panels) shows that rat brain tissue extracts elicited an increase in cytoplasmic calcium levels in HEK293 cells expressing an orphan GPCR originally termed HFGAN72. One major activity peak (designated by A) and two minor activity peaks (designated by B, BÕ) located in the HFGAN72 cells were not found in cells expressing HPRAJ70 receptors, suggesting these activity peaks were specific to the HFGAN72 receptor. After the activity peaks were localized, fractions were purified through four steps of acid-acetone extracts of rat brains by affinity chromatography. Figure 1 A (top panel), B, C and D are chromatograms showing the purification of the major activity peak, A, located in fractions 60-61. The two minor activity peaks, B and BÕ, were also purified by the same methodology (data not shown). These activity peaks appeared to be peptidic, for protease treatment of the active fractions destroyed the activity. The final purified material was structurally analyzed by limited protease digestions, nanoelectrospray tandem mass spectrometry, Edman degradation and MALDI mass spectrometry. The peptide corresponding to the major activity peak was termed Orexin-A, and was found to be a 33 amino acid peptide of 3562 Da with an N-terminal pyroglutamyl residue and C-terminal amidation as indicated by Figure 2A. Also in Figure 2A, we see the amino acid similarities of orexin-A from human and bovine origin, and the topology of the Cys disulfide bond residues. In similar methodology, the structures of purified orexin-B and orexin-BÕ, which correspond to the minor activity peaks of the HFGAN72 receptor, were determined. Orexin B contained a 28 amino acid, C-terminally amidated linear peptide of 2937 Da, while orexin BÕ contained an N-terminally truncated orexin-B. Figure 2A shows a comparison between the amino acid sequences of orexin-A and orexin-B, where black boxes represent identical residues. Sequences were found to be 46% similar. We also see amino acid residue conservation between human (83%) and mouse (95%) prepo-orexin sequences to their rat counterparts.
The next step was to describe the structure of the Prepo-Orexin precursor, for a BLAST search of the Genbank database recognized no similar sequences to orexin-A or B. cDNA encoding the precursor polypeptide was obtained by first locating a cDNA fragment encoding a part of orexin-A by RT-PCR of rat brain mRNA using primers based on parts of the known orexin-A sequence obtained earlier. Then, 5ÕRACE and 3ÓRACE reactions were performed to obtain full length cDNA. Isolated human and mouse genomic fragments containing the prepo-orexin gene were used in order to predict the sequences of the human and mouse prepo-orexin polypeptides. Figure 2B shows the deduced amino acid sequences of human, rat and mouse prepo-orexin precursor polypeptides, and demonstrates that the precursor encodes for both orexin-A and orexin-B peptides (shown in black boxes). Following the sequence analysis of the prepo-orexin polypeptide, radiation hybrid mapping showed that the human prepo-orexin gene is most tightly linked to the MIT STS markers WI-6595 and UTR9641. This location is localized on chromosome 17, at band 21 on the q arm. Interestingly, this location suggests a link between the prepo-orexin gene and a group of neurodegenerative disorders termed Òchromosome 17-lonked dementiaÓ. Only further experimentation can determine if there is such a link (Future experimentation possibilities will be discussed later).
The next step is to characterize the orexin receptor. Figure 2C shows the deduced amino acid sequences of the two similar rat and human GPCRs, now called OX1 R and OX2 R. Identical amino acid residues are boxed, and we see a high degree of similarity between these two receptors (64%), and a high degree of conservation between human and rat. When compared to other GPCRs, OX1 Ris most similar to certain neuropeptide receptors, which is supporting evidence that OX1 R is the receptor for orexins, which are in a class of small regulatory peptides.
Next, in vitro functional assays were performed to further characterize the orexin/ OX1 R interaction. Two functional tests were run: one to test binding affinity, and another to test calcium levels that could indicate protein/receptor activity. Figure 3A shows that CHO cells transfected with an expression vector containing the human OX1 R cDNA conferred the ability to bind orexin-A, while mock transfected CHO cells did not exhibit detectable levels of binding affinity (data not shown). Also, we see that orexin-B binded to the CHO/OX1 R cells, but at a significantly lower affinity. In a competitive binding assay, the IC50 for orexin-A was 20nM, while the IC50 orexin-B was 420nM, some 2-3 orders lower in magnitude. In the receptor activity functional assay shown in Figure 3C, we see that orexin-A induced a transient increase in calcium concentration in CHO/OX1 R cells, but failed to do so in mock transfected CHO cells (data not shown). Again, we see that orexin-B does induce an increase in calcium concentration, but not to the extent that orexin-A does. The EC50 in the [Ca++] transient assay for orexin-A was 30 nM, while orexin-B had a value of 2500nM. The results presented in Figures 3A and C confirm that orexin-A is indeed a specific, high affinity agonist for OX1 R, but orexin-B migh not be so specific. Because orexin-B seemed to be less specific to OX1 R, the researchers investigated the possibility that there exists some other receptor that orexin-B might bind to more specifically. A BLAST GenBank search was conducted on the OX1 R amino acid sequence, and a two highly similar sequences were found. Full length cloning and sequencing showed that this cDNA encodes for a GPCR, termed OX2 R. To functionally characterize OX2 R, tests identical to those performed on OX1 R were completed. We see in Figure 3B that CHO cells transfected with vectors containing OX2 R cDNA showed a high binding affinity for orexin-A and B, while Figure 3D shows that an increase in [Ca++ ] was induced in these cells by the addition of orexin-A and B. It appears that while the OX1 R receptor is specific for orexin-A, the OX2 R is a non selective receptor for both orexin-A and B. With these figures, I would like to have seen the negative control mock transfected CHO cells included on the figures. As the figures stand now, the reader has no visual confirmation that there was in fact a difference between the transfected and mock transfected cells. Also, another negative control, using CHO cells transfected with a vector containing the cDNA of some other known GPCR, would have been nice.
Now that the receptors and binding peptides have been described, the researchers sought to determine the location of prepo-orexin mRNA and orexin receptor mRNA. Figure 4 is a northern blot analysis of various rat tissues. The top panel shows that the orexin peptides are found only in rat brain and testis tissues; the second and third panels show that the OX1 R and OX2 R receptors are found only in rat brain tissue, while the positive control B-actin was present in all tissues, as expected. The positive control gives this figure credibility, and allows the researchers to conclude that the orexin peptidesÕ mRNA and their receptor mRNA are located almost exclusively in the brain tissues and thus function within the central nervous system. Similar analysis of human tissues was run, and similar results were found (data not shown).
Next, to further localize the orexin containing neurons in the rat brain tissue, in situ hybridization and immunohistochemical analyses were performed. In Figure 5, we see that In situ hybridization, using rat prepo-orexin cRNA as a probe, indicates orexin containing neurons organized bilaterally and symmetrically in hypothalamic and subthalamic areas of the adult rat brain, as well as positive staining in the lateral and posterior hypothalamic areas, the perifornical nucleus, subthalamus, zona incerta, subincertal and subthalamic nuclei.
Because these orexin peptides and receptors were are located in the lateral hypothalamic area , it was hypothesized that they might play a role in the regulation of feeding behavior and energy homeostasis, for previous experiments had shown that animals with lateral hypothalamic lesions had decreased food intake and lower body weight. To test orexin-A and BÕs and OX1 R and OX2 RÕs role in feeding behavior, orexin was administered acutely into the lateral ventricle of male rats through preimplanted indwelling catheters. Figure 6 shows the differential food consumption of rats with varying doses of orexin-A and B injected. These results show that both orexin-A and B stimulated increased food consumption in a dose dependent manner, with the larger doses stimulating higher food consumption. The control mice showed no such increase in food consumption. However, although both orexins stimulated increased food consumption, orexin-A had longer lasting effects than orexin-B. It is uncertain as to why these peptides differ in the duration of their effects, but future experimentation might answer this question.
To further test the role of the orexins on the regulation of feeding behavior, the researchers next performed an experiment to test the up-regulation of of prepo-orexin mRNA in the fasting state. To do this, they compared the levels of prepo-orexin mRNA in the hypothalamus of fed and fasted rats. Figure 7A is a northern blot analysis probing for propo-orexin mRNA in the hypothalamus of fed and fasted rats. We see that there are orexin bands, NPY bands, and Bactin positive control bands were present in all tissue tested. To determine if the prepo-orexin mRNA was upregulated in fasting rats as compared to fed rats, the ratio of banding intensities of orexin and NPY bands to those of the positive control bands were analyzed. Figure 7B demonstrates that the ratio of prepo-orexin mRNA to B-actin band intensities was greater in fasted rats compared to fed rats. NPY also appears to be upregulated in fasting rats.
The discovery of the orexins and their receptors, and their apparent role in feeding behavior has opened many doors for future research directions. First, although feeding behavior plays a very important role in energy homeostasis , it is by no means the only physiological process that helps the body maintain a balance of nutrients and energy expenditure. In fact, energy homeostasis is a complicated mixture of physiological processes. Therefore, one would expect there to be many peptides working together in the bodyÕs maintenance of energy homeostasis. There have already been many proteins characterized that do in fact help regulate feeding behavior. For example, NPY, a hypothalamic neuropeptide, has been established as a positive regulator of feeding behavior in mice, and MCH, another hypothalamic neuropeptide, was recently reported to stimulate food intake upon administration. To test for protein-protein interactions between the orexins, MCH and NPY and any other likely peptide (particularly one found in the hypothalamus) would provide more useful information.
To do this, I recommend one of two methods: Immunoprecipitation, or the yeast two hybrid system. For immunoprecipitation, a mouse specific anti rat mAB specific for orexin-A or orexin-B could be used to immunoprecipitate bedes with covalently linked antibody from mouse brain cells. Immunoprecipitation can separate orexin-A and B from all nonspecific proteins, but associated proteins will remain attached to the orexin proteins after the mild wash. If there are any protein-protein interactions, only the presence of the other protein and itÕs MW can be determined. If you already know the molecular weight of the associated protein (for example, if the associated protein had the same MW as that of MCH) you could conclude that this associated protein is in fact MCH or some other protein. A better method to study protein-protein interactions could be the yeast two-hybrid system, for this system not only allows you to locate an associated protein, but the genes that encode this protein can be cloned for further study.
To do this, two hybrids should be constructed and expressed in plasmids: one that consists of the DNA binding domain of the yeast transcriptional activator protein GAL4 fused to orexin; the other hybrid consisting of the GAL4 activation domain protein sequences encoded by a library of mouse, human or rat genomic DNA. If any proteins interact with the orexins, we have the immediate availability of the cloned gene for the interacting protein.
In either of these methods to test for interactions with orexin, we would be looking for the MCH and NPY proteins, for they have all been shown to be involved in feeding behavior, but a close lookout for new proteins would important. Because energy homeostasis is so complex, we would expect to find some new proteins that also play a role in energy homeostasis. With the identification of new proteins, we could duplicate this experimentÕs methodology to locate and structurally describe the proteins and their receptors.
OVERALL OPINION ON THE PAPER:
I was very impressed by the researchers thorough treatment of the data and the logical line of experimentation to further convince the reader of their conclusions as well as test new hypotheses along the way. A thorough analysis of the structure of the orexins and their receptors, coupled with functional assays to support the protein/receptor activity showed clear evidence that the protein and receptor interacted. An investigation of location in brain tissues, followed by more functional experiments directly related to feeding behavior provided clear evidence that these orexins and their receptors played some role in feeding behavior.
Reference: Sakurai et al. 1998. "Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feeding Behavior". Cell, Vol. 92, 573-585.
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