DEFINITION
Galanin is an endocrin neuropeptide of 29-30 (30 in human) aa, encoded by the GAL gene. Galanin was first identified from porcine intestinal extracts in 1983 (Galanin- a novel biologically active peptide from porcine intestine, 1983) and it is widely expressed in brain, spinal cord, and gut of humans as well as other mammals. Galanin signaling occurs through three G protein-coupled receptors (GalR1, GalR2, GalR3).
His best-known function is the modulation and inhibition of action potentials in neurons, predominantly is an hyperpolarizing neuropeptide.
His name derives from a N-terminal glycine residue and a C-terminal alanine.
THE GENE
Galanin in synthetized primarly as preprogalanin by the GAL gene. An approximately 35-kb region of genomic DNA encoding the human preprogalanin gene including 5′ and 3′ flanking sequences has been cloned and characterized. Exons and flanking introns were sequenced to determine the structural organization of the gene. The gene spans 6.5 kb, with the first exon encoding only the 5′ untranslated sequence. The coding region of preprogalanin and the 3′ untranslated sequence is divided into five exons. The position of the single human preprogalanin gene was localized to chromosome 11q13.3–q13.5. Several oncogenes have been mapped to this region, which is also the breakpoint for the translocation t(11;14)(q13;q32) in chronic lymphocytic leukemia and diffuse B-cell lymphoma
CHEMICAL STRUCTURE AND IMAGES
The sequence of this gene is highly conserved among mammals, showing over 85% homology between rat, mouse, porcine, bovine, and human sequences. In these animal forms, the first 15 amino acids from the N-terminus are identical, but amino acids differ at several positions on the C-terminal end of the protein.
These slight differences in protein structure have far-reaching implications on their function. For example, porcine and rat galanin inhibit glucose-induced insulin secretion in rats and dogs but have no effect on insulin secretion in humans. This demonstrates that it is essential to study the effects of galanin, and other regulatory peptides, in their autologous species.
Protein Aminoacids Percentage
Protein Aminoacids Percentage (Width 700 px)
GALANIN
GALANIN RECEPTOR
No significant differences were observed between species in the three different Receptors, however there are some differences in Aminoacids Percentage in Human's one
SYNTHESIS AND TURNOVER
Galanin is a peptide consisting of a chain of 29 amino acids (30 amino acids in humans) produced from the cleavage of a 123-amino acid protein known as preprogalanin, which is encoded by the GAL gene
The mRNA encoding the Galanin is composed of a 5nt portion encoding a signal sequence, followed by a Lys-Arg cleavage site, then the 29-amino-acid-long galanin peptide followed by Gly-Lys-Arg at the C-terminal containing the amide donor Gly and the cleavage site Lys-Arg. Both the signal sequence and the galanin sequence show a very high degree of homology (>85%) between the rat, mouse, porcine, bovine, and human sequences.
The Galanin family of protein consists of four proteins, of which GAL was the first to be identified. The second was galanin message-associated protein (GMAP), a 59- or 60-amino acid peptide also formed from the cleavage of preprogalanin
The other two peptides, galanin-like peptide (GALP) and alarin, were identified relatively recently and are both encoded for in the same gene, the preproGALP gene. GALP and alarin are produced by different post-translational splicing of this gene
Organization of the preprogalanin gene (modified from Kofler et al., 1996). The first exon encodes only the 5′-untranslated region of preprogalanin mRNA. Exon 2 starts with the translation initiation codon of the signal peptide and terminates before the proteolytic site preceding the mature galanin peptide. The first 13 amino acids of galanin are encoded by exon 3; the remaining 16 amino acids and most of GMAP by exons 4 and 5. The remaining portion of GMAP and the polyadenylation site are located in exon 6. Arrows indicate cleavage sites of endopeptidases.
Organization of the preproGALP gene. The first exon is noncoding. PreproGALP is encoded by exons 2–6 and the segment with galanin homology [(GALP (9–21)] is contained in exon 3. The mature peptide GALP (1–60) is encoded by exons 2–5. Post-transcriptional splicing leads to exclusion of exon 3 resulting in a frame shift and a novel precursor protein. This protein harbors the signal sequence of preproGALP and the first 5 amino acids of the mature GALP peptide followed by another 20 amino acids and proteolytic cleavage leads to alarin (1–25). Arrows indicate potential cleavage sites of endopeptidases
FUNCTIONS
Thanks to his role of hyperpolarization and neuromodulatory peptide, Galanin is implicated in different physiological and pathological meccanism
Signaling pathways of galanin receptor subtypes. Abbreviations: AC, adenylate cyclase; CaCC, Ca2+-dependent chloride channel; cAMP, 3′,5′-cyclic adenosine monophosphate; (p)CREB, (phosphorylated) cAMP response element binding protein; 3′,5′-cAMP response element-binding protein; DAG, diacylglycerol; IP3, inositol triphosphate; MEK, mitogen-induced extracellular kinase; PDK-1, phosphoinosotide-dependent protein-kinase 1; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol trisphosphate; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PLC, phospholipase C
Feeding and Metabolism
Initially was observed that Galanin and biologically active fragments such as galanin (1–16) stimulate food intake following acute central injection into the hypothalamic paraventricular, lateral and ventromedial nuclei, and the central nucleus of the amygdala, producing a rapid increase in the feeding response and total caloric intake without altering feeding-associated behaviors such as drinking, grooming, and motor activity.
However, a recent detailed study of GalR1-KO mice fed diets containing differing levels of energy and fat, concluded that the endogenous galanin-GalR1 system does play a significant role in adjusting food intake and/or metabolism to acute changes in dietary fat. In response to an acute 3-day high-fat challenge, GalR1-KO mice displayed an impaired adaptation, leading to increased food intake and weight gain, compared to normal food intake and weight modulation on low-fat diets. This latter finding is consistent with the phenotype reported for the galanin-KO, which is also more sensitive to leptin treatment. In contrast to this acute response, over a subsequent 2-week period on the high-fat diet, GalR1-KO mice consumed less food and daily energy than when maintained on a low-fat diet and less food and energy than their heterozygous littermates, suggesting that overall, GalR1 may oppose positive energy balance or help maintain neutral balance.
Adaptation to high-fat /high-energy diets in age-matched, adult, male GALR1 knockout (−/−; KO, n = 5), GALR1 heterozygote (+/−; hetero, n = 7), and wild-type (+/+; C57BL/6J, n = 5) mice. Data reflect mean ± S.E.M. (top) daily food intake, (middle) daily weight gain, and (bottom) feed efficiency in mice while maintained for 10 days on purified low-fat/low-energy control diet or during a 3-day “challenge” of being switched to high-fat/high-energy diet. p < 0.05 vs. respective wild-type control, #p < 0.05 vs. respective low-fat diet condition (Fisher's LSD tests).
Osmotic regulation and water intake
Early studies of galanin dynamics in hypothalamic vasopressin neurons and the effects of central galanin administration revealed that galanin is involved in osmotic regulation at the level of the hypothalamus. Vasopressin is pivotally involved in osmotic regulation, and vasopressin-deficient and salt-loaded rats with increased plasma osmolarity have reduced concentrations of galanin in the neurointermediate lobe of the pituitary and in the median eminence.(Potential involvement of galanin in the regulation of fluid homeostasis in the rat, 1989)
Salt loading and dehydration increase both vasopressin release and the level of galanin; the latter in turn acts as a negative feedback modulator of vasopressin release.
The subfornical organ (SFO) is a circumventricular structure that plays an important role in the control of water intake and vasopressin release, and a recent study demonstrated galanin-positive synapses in the SFO and investigated a role for galanin in regulating the activity of SFO neurons in vitro. In SFO slice preparations, galanin dose-dependently inhibited the neural activity of SFO neuron
The GalR1 agonist, M617 also inhibited SFO cells, whereas the GalR2/3 agonist galanin (2–11) had no effect, suggesting that galanin responses were largely mediated by GalR1.
Inhibitory effects of GAL on spontaneous neural activities. Horizontal bars indicate drug application time, and the concentrations of GAL are indicated above the bars. (A) GAL inhibited the spontaneous electrical activity in a multi-unit recording (Aa) and a single-unit (Ab) that was discriminated from the multi-unit in Aa in dose-dependent manner
Nociception
Experimental evidence is available for galanin acting as both an inhibitory and excitatory (i.e., analgesic and hyperalgesic) mediator and the relative pro- or anti-nociceptive action of galanin appears to depend on the acute or chronic state of the nociceptive stimulus, the nature of the stimulus (thermal, mechanical or chemical), and on the concentration of galanin available to act on the nociceptive afferent nerves
The role of galanin in acute nociception has also been assessed in transgenic mice..
Intact adult galanin-KO mice have greater sensitivity to acute mechanical and thermal pain, while galanin-overexpressing (OE) mice have reduced responses to acute nociceptive heat.
In pathological, chronic pain conditions such as neuropathic pain and inflammation, expression of galanin (and several other peptides) is markedly increased in nociceptive pathways in DRG and the spinal cord.
Galanin-KO mice displayed a reduced pain response and autotomy in chronic pain models.
On the basis of numerous data, it was suggested that GalR2 may mediate the hyperalgesic response in chronic pain, while GalR1 mediates the analgesic effect (Nerve injury induces plasticity that results in spinal inhibitory effects of galanin, 2002)
Learning and Memory
The effect of galanin on learning and memory has also been studied in 2 types of galanin-OE mice. In one strain the galanin gene is linked to the dopamine-β-hydroxylase (D-β-H) promoter, restricting galanin overexpression to (nor)adrenergic neurons; and in a second strain, the galanin gene is linked to the platelet-derived growth factor-β (PDGF) promoter, producing a wider distribution and higher levels (4- to 8-fold) of galanin expression in the hippocampus and cortex. D-β-H galanin-OE mice displayed deficits in learning and memory tests such as spatial navigation and olfactory- and emotional memories associated with attenuated depolarization-induced glutamate release from hippocampal neurons, while measures of sensory and motor abilities and levels of extracellular noradrenaline or serotonin release in the hippocampus were normal. (Modulation of hippocampal excitability and seizures by galanin, 2000)
In association with inhibition of cognitive performance, galanin has also been shown to reduce long-term potentiation (LTP) of hippocampal CA1 neuron activity in rodents and a recent study examined the role of GalR1 and GalR2 in these actions
Gastrointestinal tract
Since galanin was originally isolated from porcine small intestine, the GI tract has subsequently been investigated extensively in an effort to uncover possible physiological functions of galanin. Galanin was found to be widely distributed throughout the digestive tract with galanin-LI present in enteric neurons and in nerve fibers projecting to all layers of the GI tract wall of all mammalian species investigated, including human.
Galanin was also found to exert multiple biological effects in the GI tract. It inhibits gastric acid secretion and the release of numerous pancreatic peptides, including insulin, amylase, glucagon, and somatostatin. Furthermore, galanin modulates GI motility, either via a direct action on intestinal smooth muscle cells or by indirect neuromodulatory mechanisms, such as the release of other transmitters. (Antisecretory effects of galanin and its putative antagonists M15, M35 and C7 in the rat stomach, 2000)
The effects of galanin on GI motility are both stimulatory and inhibitory and depend on the species and the GI tract segment investigated
Pathology
Alzheimer's disease
In post-mortem brains from Alzheimer's victims, an increase of galanin receptor binding sites of up to 200% was observed in several layers of the hippocampal CA1 region, the stratum radiatum of CA3, the hilus of the dentate gyrus and the substantia nigra.
Increased galanin receptor expression is also evident in the central nucleus of the amygdala and the cortico-amygdaloid transition area in the early stages of Alzheimer's disease, but levels decrease towards the end-stages. (Galanin receptor over-expression within the amygdala in early Alzheimer's disease: An in vitro autoradiographic analysis, 2002) Galanin-positive fibers and terminals are present in a higher density in the basal forebrain and they hyper-innervate the remaining cholinergic cell bodies.
It has been proposed that the inhibitory activity of galanin might inhibit ACh release and worsen symptoms; although more recent findings indicate otherwise. In electrophysiological studies of acutely dissociated rat cholinergic neurons from basal forebrain, galanin inhibited K+ currents, but not Ca2+ or Na+ currents.
It is still unclear whether upregulation of galanin in Alzheimer's is a contributing factor to the disease, or a result of nerve injury, or a compensatory change to maintain cholinergic transmission.
Diabetes
Galanin has been detected in pancreatic islet cells and nerves in several species. A large number of galanin-like immunoreactive cells were observed in both the peripheral and central regions of the islet of Langerhans of normal rat pancreas. In contrast, pancreatic islets in diabetic rats contained significantly less galanin-LI cells, because galanin is synthesized by insulin producing cells that are reduced in these animals. Galanin has an inhibitory effect on insulin secretion from the isolated pancreas of normal and diabetic rats and this presumably represents an autocrine action of the peptide, as galanin co-localizes with insulin.
Galanin has actions at several levels in the complex regulatory circuits controlling glucose metabolism and feeding: at the hypothalamic level regulating food intake and dietary preference, and in the pancreas, regulating sympathetic nerve activity and islet cell secretion (an autocrine action). Further studies are necessary to elaborate the exact sites of action of galanin in feeding and glucose homeostasis, the relative contribution of central (e.g. hypothalamic) versus peripheral (e.g. pancreatic) galanin, and the receptor subtypes involved.
In humans, galanin injection has been shown to suppress the initial postprandial rise in the plasma concentration of glucose and insulin with an unaltered glucose-stimulated insulin release (Inhibitory effect of galanin on postprandial gastrointestinal motility and gut hormone release in humans, 1989) and in a cohort of 91 children, plasma levels of galanin were found to be higher among children with insulin-dependent diabetes mellitus compared to healthy subjects.
In patients with type II diabetes mellitus, therefore, a strong positive correlation of the galanin plasma concentration with glucose in the fasting state was observed in both sexes.
Cancer
The first evidence that the galanin peptide family might be involved in the pathophysiology of neoplasms came from studies on human tumors arising from neuroendocrine cells such as adrenal pheochromocytomas, in which increased galanin-LI was detected.
Numerous studies have observed galanin-LI in human pituitary adenomas.
Galanin-LI and galanin mRNA expression have also been detected in human pediatric neuroblastic tumors and in 18 out of 20 human gliomas without significant clinical relevance.
Prostate carcinomas also displayed galanin binding sites as other human cancers, whereas other tumors such as renal cell carcinonoma, bladder carcinoma, Wilm's tumor, rhabdomyaosarcoma, Ewing sarcoma, Hodgkin's lymphoma and squamous epithelial carcinoma exhibited no galanin binding.
Contradictory roles for galanin and its receptors in various tumors have been reported so in order to understand their function, investigations of individual galanin receptors are necessary. In head and neck squamous carcinoma cells (HNSCC) with silenced GalR1 and GalR2, is showned that reexpressed GalR1 suppresses tumor cell proliferation via Erk1/2-mediated effects on cdk inhibitors and cyclin D1. Others showed that GAal2 could induce apoptosis in neuroblastoma cells with wild-type p53, whereas GalR2 stimulated proliferation in small cell lung cancer. In this study, we investigated the role of GalR2 in HNSCC cells that have mutant p53 and do not express GalR1.
Using a human oral carcinoma cell line with a splice site mutation causing a 46-bp p53 off-frame deletion, stably transfected to express GalR2 (UM-SCC-1-GalR2) a Galanin treatment of this cells caused morphologic changes and a marked decrease in cell number that were not observed in UM-SCC-1-mock cells. Galanin and GalR2 resulted in decreased bromodeoxyuridine incorporation, p27Kip1 and p57Kip2 up-regulation, and decreased cyclin D1 expression. These effects were similar to GalR1 signaling in HNSCC, but GalR2 also induced caspase-3–dependent apoptosis, which was confirmed by Annexin-V staining and DNA fragmentation analysis. These were not observed with GalR1 (Galanin receptor subtype 2 suppresses cell proliferation and induces apoptosis in p53 mutant head and neck cancer cells, 2009)
Interestingly, one copy of the galanin receptor 1 (GALR1) locus on 18q is often deleted and expression is absent in some head and neck squamous cell carcinoma (HNSCC) cell lines. To determine if loss of heterozygosity and hypermethylation might silence the GalR1 gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors.
In a recent study was observed that GalR1 promoter was fully or partially methylated in 38 of 72 (52.7%) HNSCC cell lines but not in the majority 18 of 20 (90.0%) of nonmalignant lines. GalR1 methylation was also found in 38 of 100 (38%) primary tumor specimens. Methylation correlated with decreased GalR1 expression. In tumors, methylation was significantly correlated with increased tumor size, lymph node status, tumor stage, cyclin D1 expression, and p16 methylation and survival. Bisulfite sequencing of 36 CpG sites upstream of the transcription start site revealed that CpG methylation within transcription factor binding sites correlated with complete suppression of GalR1 mRNA. Treatment with trichostatin A and 5-azacytidine restored GalR1 expression. In UM-SCC-23 cells that have total silencing of GalR1, exogenous GalR1 expression and stimulation with galanin suppressed cell proliferation. (Epigenetic inactivation of galanin receptor 1 in head and neck cance, 2008)
The expression of galanin and its receptors supports a role for galanin in tumor cell pathology via autocrine/paracrine mechanisms, with stimulating or antiproliferation actions depends on neoplasm's cell origins, with potential implications for diagnosis and treatment of cancer.
