Jesus P. Camiña

Obestatin‐mediated proliferation of human retinal pigment epithelial cells: Regulatory mechanisms

In this work, we have evaluated the effect of the new discovered peptide obestatin on cell proliferation in primary cultures of human retinal epithelial cells (hRPE cells). The results showed that this peptide induced, in a dose‐dependent manner, cell proliferation by MEK/ERK 1/2 phosphorylation. A sequential analysis of the obestatin transmembrane signaling pathway showed that the ERK 1/2 activity is partially blocked after preincubation of the cells with pertussis toxin (PTX), as well as by wortmannin (an inhibitor of PI3K), claphostin C (an inhibitor of PKC), and PP2 (which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho‐PKCε‐, θ‐, and µ‐isoenzymes rise with different time courses, from which PKCε might be responsible for ERK 1/2 response. Based on the experimental data, a signaling pathway involving the consecutive activation of Gi, PI3K, novel PKC (probably PKCε), and Src for ERK 1/2 activation is proposed. These results incorporate a new mitogenic factor to the group of factors that regulate proliferation of hRPE cells. J. Cell. Physiol. 211: 1–9, 2007.

Agonist-Specific Coupling of Growth Hormone Secretagogue Receptor Type 1a to Different Intracellular Signaling Systems

The growth hormone secretagogue receptor subtype 1a (GHSR-1a) is involved in biological actions of ghrelin by triggering intracellular second messengers coupled to heterotrimeric G-protein complex involving Gαq/11. Adenosine is a partial agonist of the GHSR-1a, binding to a binding pocket distinct from the one described for ghrelin. This suggests a variety of functions for the poorly understood GHSR1a receptor. In this work, a sequential analysis of the pathways involved in the regulation of GHSR-1a signaling was undertaken to characterize the intracellular calcium mobilization that is observed following adenosine binding. The results showed that adenosine induced, in a dose-dependent manner, a calcium mobilization from IP3-sensitive intracellular stores since the IP3 receptor blocker 2-APB was able to suppress the calcium response. However, adenosine did not show any effect in the formation of inositol phosphates. The calcium-mobilizing activity was blocked after preincubation of cells with CTX, the inhibitor of adenylate cyclase MDL-12,330A and the protein kinase A blocker H-89. Furthermore, the administration of adenosine stimulated cAMP production. Based on the experimental data, a signaling pathway is proposed involving adenylate cyclase and protein kinase A, which causes phosphorylation of the IP3 receptor, with a cross-talk between the signaling pathways activated by ghrelin and adenosine. The data described in this report suggest that GHSR-1a is able to activate different intracellular second-messenger systems depending on the agonist that activates it. The regulation of the ghrelin-activated earliest signaling pathways by adenosine may have unexpected implications in the GHSR-1a actions.

Regulation of ghrelin secretion and action

The pulsatile release of growth hormone (GH) from anterior pituitary gland is regulated by the interplay of at least two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph. Furthermore, release of GH in vivo may also be controlled by a third type of receptor, the growth hormone secretagogue receptor, a G-protein-coupled receptor, called GHS receptor type 1a (GHS-R1a), which was identified in the pituitary and the hypothalamus in humans using a nonpeptidyl growth hormone secretagogue (MK-0677). Ghrelin, the endogenous ligand for the GHS-R1a, is a 28-amino-acid peptide isolated from human stomach that is modified by a straight chain octanoyl group covalently linked to Ser3, which is essential for its endocrine activity. This hormone, predominantly expressed and secreted by the stomach, has a dual action on GH secretion and food intake, showing interdependency between these actions. The finding that fasting and food intake, respectively, increase and decrease the secretion of ghrelin suggests that this hormone may be the bridge connecting somatic growth and body composition with energy metabolism, and appears to play a role in the alteration of energy homeostasis and body weight in pathophysiological states such as hypothyroidism and hyperthyroidism. Despite this, little is known about the intracellular signaling through which ghrelin exerts its regulatory actions. Activation of intracellular calcium mobilization is one of the earliest known cellular signals elicited by ghrelin. In HEK-293 cells expressing the GHS-R1a, ghrelin induces a biphasic cytosolic calcium elevation characterized by a spike phase of the response, which reflects Ins(1,4,5)P3-dependent calcium mobilization of intracellular stores, and a sustained phase of the response, which is due to calcium influx across the plasma membrane triggered by aperture of capacitative calcium channels (store-operated calcium channels). Upon repeated administration, ghrelin showed a marked suppression of ghrelin-mediated elevations of intracellular calcium. This homologous desensitization represents an important physiological mechanism that modulates receptor responsiveness and acts as an information filter for intracellular signaling system. The discovery of ghrelin adds a new component to the complex machinery responsible for regulation of GH secretion in connection with the regulation of appetite and energy homeostasis.

Stimulation by ghrelin of p42/p44 mitogen-activated protein kinase through the GHS-R1a receptor : Role of G-proteins and β-arrestins

Results presented in this study indicate that in human embryonic kidney 293 cells (HEK 293), the ghrelin receptor growth hormone secretagogue receptor type 1a (GHS‐R1a) activates the extracellular signal‐related kinases 1 and 2 (ERK 1/2) via three pathways. One pathway is mediated by the β‐arrestins 1 and 2, and requires entry of the receptor into a multiprotein complex with the β‐arrestins, Src, Raf‐1, and ERK 1/2. A second pathway is Gq/11‐dependent and involves a Ca2+‐dependent PKC (PKCα/β) and Src. A third pathway is Gi‐dependent and involves phosphoinositide 3‐kinase (PI3K), PKCε, and Src. Our current study reveals that Gi/o‐ and Gq/11‐proteins are crucially involved in the β‐arrestin‐mediated ERK 1/2 activation. These results thus support the view that the β‐arrestins act as both scaffolding proteins and signal transducers in ERK 1/2 activation, as reported for other receptors. The different pathways of ERK 1/2 activation suggest that binding to GHS‐R1a activates ERK 1/2 pools at different locations within the cell, and thus probably with different physiological consequences. J. Cell. Physiol. 213: 187–200, 2007.

cis-Unsaturated Free Fatty Acids Block Growth Hormone and Prolactin Secretion in Thyrotropin-Releasing Hormone-Stimulated GH3 Cells by Perturbing the Function of Plasma Membrane Integral Proteins

In vivo FFA block basal and stimulated GH secretion and have been implicated in the pathogenesis of the altered GH secretion present in obesity and Cushing’s syndrome. Although a direct action on the somatotroph cell has been postulated, the FFA mechanism of action is unknown. The main biological target for FFA action is the cellular membrane, and it has been shown that these metabolites can block the activity of a number of plasma membrane pumps, channels, and receptor systems. In the present work, it was observed using different types of pituitary cells (GH3, GH4C1, and rat pituitary primary cultures) that cis-unsaturated fatty acids, such as oleic, 1) do not perturb TRH binding or the homologous desensitization of the TRH receptor; 2) inhibit TRH-induced inositol 1,4,5-trisphosphate/diacylglycerol generation, probably by a direct perturbation of phospholipase C; 3) reduce the TRH-induced intracellular Ca2+ redistribution and the ensuing changes in membrane potential; 4) completely inhibit the[ Ca2+]i r...

Obestatin stimulates Akt signalling in gastric cancer cells through β-arrestin-mediated epidermal growth factor receptor transactivation

Obestatin was identified as a gut peptide encoded by the ghrelin gene that interacts with the G protein-coupled receptor, GPR39. In this work, a sequential analysis of its transmembrane signalling pathway has been undertaken to characterize the intracellular mechanisms responsible for Akt activation. The results show that Akt activation requires the phosphorylation of T308 in the A-loop by the phosphoinositide-dependent kinase 1 (PDK1) and S473 within the HM by the mammalian target of rapamycin (mTOR) kinase complex 2 (mTORC2: Rictor, mLST8, mSin1, mTOR kinase) with participation neither of G(i)(/o)-protein nor Gbetagamma dimers. Obestatin induces the association of GPR39/beta-arrestin 1/Src signalling complex resulting in the transactivation of the epidermal growth factor receptor (EGFR) and downstream Akt signalling. Upon administration of obestatin, phosphorylation of mTOR (S2448) and p70S6K1 (T389) rise with a time course that parallels that of Akt activation. Based on the experimental data obtained, a signalling pathway involving a beta-arrestin 1 scaffolding complex and EGFR to activate Akt signalling is proposed.

Evidence of free leptin in human seminal plasma

Leptin is an adipose tissue-secreted hormone that actively participates in the regulation of energy homeostasis. Besides this principal role, leptin has been implicated in a large variety of neuroendocrine, paracrine, and autocrine actions involved in the regulation of reproductive function in both experimental animals and humans. Although the participation of leptin in female reproduction is well established, any role in male reproductive function is at best tenuous. The aim of this study was to ascertain whether true leptin is present in human seminal fluid and the tissue of its production. Pooled human seminal plasma obtained from healthy donors showed by direct radioimmunoassay (RIA) the presence of radioimmunoassayable leptin. Serial dilutions of unextracted semen paralleled the RIA standard curve, also devoid of interference in the assay. To prove that this activity was true leptin, seminal plasma was subjected to size-exclusion chromatography, which showed that leptin immunoreactivity eluted with the same partition coefficient as cold leptin, 125I-leptin, and 125I-leptin preincubated with seminal plasma. The results demonstrate that true leptin was present in semen in a free form, i.e., without binding proteins. The presence of leptin charge variants in seminal plasma was assessed by anion-exchange chromatography, which showed two peaks of leptin inmunoreactivity, while 125I-leptin eluted as a single peak. Preincubation of 125I-leptin with seminal fluid converted the single peak into a double peak, indicating that components of the seminal fluid introduce a charge variation in leptin. Leptin levels in seminal plasma of 40 healthy men were 0.95±0.19 ng/mL while in 5 vasectomized men the levels were 0.92±0.25 ng/mL, suggesting that testicular tissues were not the source of seminal leptin. No correlation was observed between leptin concentrations in semen and the physical characteristics of semen samples or physical characteristics of spermatozoids, such as concentration, motility, vitality, or morphology. In conclusion it was unambiguously demonstrated that human leptin is present in seminal fluid, with at least two charge variants and no binding proteins, the most likely source being either seminal vesicles or prostate tissue. The role of seminal fluid leptin in the male reproductive function or sperm capacitation is at present unknown.

Growth hormone secretagogues as diagnostic tools in disease states

One of the most active topics in the growth hormone-IGF-1 field is that of the so-called growth hormone secretagogues (GHS). At a time when the isolation of GHRH had not occurred, the GHS were developed as artificial tools to release GH. The interest in these groups of compounds was rekindled when it was realized that they were not surrogates of GHRH nor were they acting through the modulation of the release of either GHRH or somatostatin. With the subsequent cloning of the specific receptor of GHS, and today of the natural ligand for that receptor, named ghrelin, it soon become clear that GHS and the GHS-receptor were part of a new physiological system involved in GH regulation. The dual control of GH secretion became a trinity. GHS releases GH when administered by any route—oral, iv, sc, and even transdermally—with a surprising potency and reproductivity. In addition, GHS when administered together with GHRH exert a synergistic action on GH secretion and that combined administration is the most potent GH releaser to date. Clinical studies have demonstrated that the GHS-GHRH administration may be considered the new “gold standard” test of GH reserve in humans, as the GH secretion so elicited is not altered by gender, adiposity, or age. The combined administration of GHRH plus GHS is able to discriminate between healthy subjects and patients with adult GH deficiency, suggesting a considerable utility in the clinical setting.

The Obestatin/GPR39 System Is Up-regulated by Muscle Injury and Functions as an Autocrine Regenerative System

Background: Satellite cell activation is orchestrated by several signals, which induce their differentiation into skeletal muscle fibers. Results: Obestatin and the GPR39 receptor exert an autocrine role on the control of myogenesis. Conclusion: Our data indicate that obestatin/GPR39 is an injury-regulated signal that functions as a myogenic regenerative system. Significance: Strategies to enhance obestatin-mediated signaling could be useful in treating trauma-induced muscle injuries and skeletal muscle myopathies. The maintenance and repair of skeletal muscle are attributable to an elaborate interaction between extrinsic and intrinsic regulatory signals that regulate the myogenic process. In the present work, we showed that obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor are expressed in rat skeletal muscle and are up-regulated upon experimental injury. To define their roles in muscle regeneration, L6E9 cells were used to perform in vitro assays. For the in vivo assays, skeletal muscle tissue was obtained from male rats and maintained under continuous subcutaneous infusion of obestatin. In differentiating L6E9 cells, preproghrelin expression and correspondingly obestatin increased during myogenesis being sustained throughout terminal differentiation. Autocrine action was demonstrated by neutralization of the endogenous obestatin secreted by differentiating L6E9 cells using a specific anti-obestatin antibody. Knockdown experiments by preproghrelin siRNA confirmed the contribution of obestatin to the myogenic program. Furthermore, GPR39 siRNA reduced obestatin action and myogenic differentiation. Exogenous obestatin stimulation was also shown to regulate myoblast migration and proliferation. Furthermore, the addition of obestatin to the differentiation medium increased myogenic differentiation of L6E9 cells. The relevance of the actions of obestatin was confirmed in vivo by the up-regulation of Pax-7, MyoD, Myf5, Myf6, myogenin, and myosin heavy chain (MHC) in obestatin-infused rats when compared with saline-infused rats. These data elucidate a novel mechanism whereby the obestatin/GPR39 system is coordinately regulated as part of the myogenic program and operates as an autocrine signal regulating skeletal myogenesis.

Preproghrelin expression is a key target for insulin action on adipogenesis

This study aimed to investigate the role of preproghrelin-derived peptides in adipogenesis. Immunocytochemical analysis of 3T3-L1 adipocyte cells showed stronger preproghrelin expression compared with that observed in 3T3-L1 preadipocyte cells. Insulin promoted this expression throughout adipogenesis identifying mTORC1 as a critical downstream substrate for this profile. The role of preproghrelin-derived peptides on the differentiation process was supported by preproghrelin knockdown experiments, which revealed its contribution to adipogenesis. Neutralization of endogenous O-acyl ghrelin (acylated ghrelin), unacylated ghrelin, and obestatin by specific antibodies supported their adipogenic potential. Furthermore, a parallel increase in the expression of ghrelin-associated enzymatic machinery, prohormone convertase 1/3 (PC1/3) and membrane-bound O-acyltransferase 4 (MBOAT4), was dependent on the expression of preproghrelin in the course of insulin-induced adipogenesis. The coexpression of preproghrelin system and their receptors, GHSR1a and GPR39, during adipogenesis supports an autocrine/paracrine role for these peptides. Preproghrelin, PC1/3, and MBOAT4 exhibited dissimilar expression depending on the white fat depot, revealing their regulation in a positive energy balance situation in mice. The results underscore a key role for preproghrelin-derived peptides on adipogenesis through an autocrine/paracrine mechanism.