José Santos-Alvarez

Role of leptin as an immunomodulator of blood mononuclear cells: mechanisms of action

Leptin is a an adipocyte‐secreted hormone that regulates weight centrally. However, the leptin receptor is expressed not only in the central nervous system, but also in peripheral tissues, such as haematopoietic and immune systems. Therefore, the physiological role of leptin should not be limited to the regulation of food intake and energy expenditure. Moreover, the leptin receptor bears homology to members of the class I cytokine family, and recent data have demonstrated that leptin is able to modulate the immune response. Thus, the leptin receptor is expressed in human peripheral blood mononuclear cells, mediating the leptin effect on proliferation and activation. In vitro activation and HIV infection in vivo induce the expression of the long isoform of the leptin receptor in mononuclear cells. Also, leptin stimulates the production of proinflammatory cytokines from cultured monocytes and enhances the production of Th1 type cytokines from stimulated lymphocytes. Moreover, leptin has a trophic effect on monocytes, preventing apoptosis induced by serum deprivation. Leptin stimulation activates JAK–STAT, IRS‐1‐PI3K and MAPK signalling pathways. Leptin also stimulates Tyr‐phosphorylation of the RNA‐binding protein Sam68 mediating the dissociation from RNA. In this way, leptin signalling could modulate RNA metabolism. These signal transduction pathways provide possible mechanisms whereby leptin may modulate activation of peripheral blood mononuclear cells. Therefore, these data support the hypothesis regarding leptin as a proinflammatory cytokine with a possible role as a link between the nutritional status and the immune response. Moreover, these immunoregulatory functions of leptin could have some relevance in the pathophysiology of obesity.

Role of Leptin in the Activation of Immune Cells

Adipose tissue is an active endocrine organ that secretes various humoral factors (adipokines), and its shift to production of proinflammatory cytokines in obesity likely contributes to the low-level systemic inflammation that may be present in metabolic syndrome-associated chronic pathologies such as atherosclerosis. Leptin is one of the most important hormones secreted by adipocytes, with a variety of physiological roles related to the control of metabolism and energy homeostasis. One of these functions is the connection between nutritional status and immune competence. The adipocyte-derived hormone leptin has been shown to regulate the immune response, innate and adaptive response, both in normal and pathological conditions. The role of leptin in regulating immune response has been assessed in vitro as well as in clinical studies. It has been shown that conditions of reduced leptin production are associated with increased infection susceptibility. Conversely, immune-mediated disorders such as autoimmune diseases are associated with increased secretion of leptin and production of proinflammatory pathogenic cytokines. Thus, leptin is a mediator of the inflammatory response.

Metabolic effects and mechanism of action of the chromogranin A-derived peptide pancreastatin.

Pancreastatin is one of the regulatory peptides derived from intracellular and/or extracellular processing of chromogranin A, the soluble acidic protein present in the secretory granules of the neuroendocrine system. While the intracellular functions of chromogranin A include formation and maturation of the secretory granule, the major extracellular functions are generation of biologically active peptides with demonstrated autocrine, paracrine or endocrine activities. In this review, we will focus on the metabolic function of one of these peptides, pancreastatin, and the mechanisms underlying its effects. Many different reported effects have implicated PST in the modulation of energy metabolism, with a general counterregulatory effect to that of insulin. Pancreastatin induces glycogenolysis in liver and lipolysis in adipocytes. Metabolic effects have been confirmed in humans. Moreover, naturally occurring human variants have been found, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptides potency to inhibit cellular glucose uptake. Thus, qualitative hereditary alterations in pancreastatins primary structure may give rise to interindividual differences in glucose and lipid metabolism. Pancreastatin activates a receptor signaling system that belongs to the seven-spanning transmembrane receptor coupled to a Gq-PLCbeta-calcium-PKC signaling pathway. Increased pancreastatin plasma levels, correlating with catecholamines levels, have been found in insulin resistance states, such as gestational diabetes or essential hypertension. Pancreastatin plays important physiological role in potentiating the metabolic effects of catecholamines, and may also play a pathophysiological role in insulin resistance states with increased sympathetic activity.

Characterization of pancreastatin receptors and signaling in adipocyte membranes

Pancreastatin (PST), a chromogranin A derived peptide with an array of effects in different tissues, has a role as a counterregulatory hormone of insulin action in hepatocytes and adipocytes, regulating glucose, lipid and protein metabolism. We have previously characterized PST receptors and signaling in rat hepatocytes, in which PST functions as a calcium-mobilizing hormone. In the present work we have studied PST receptors as well as the signal transduction pathways generated upon PST binding in adipocyte membranes. First, we have characterized PST receptors using radiolabeled PST as a ligand. Analysis of binding data indicated the existence of one class of binding sites, with a B(max) of 5 fmol/mg of protein and a K(d) of 1 nM. In addition, we have studied the G protein system that couples the PST receptor by gamma-(35)S-GTP binding studies. We have found that two G protein systems are involved, pertussis toxin-sensitive and -insensitive respectively. Specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Galpha(q/11) and to a lesser extent Galpha(i1,2) are activated by PST in rat adipocyte membranes. On the other hand, adenylate cyclase activity was not affected by PST. Finally, we have studied the specific phospholipase C isoform that is activated in response to PST. We have found that PST receptor is coupled to PLC-beta(3) via Galpha(q/11) activation in adipocyte membranes.

G protein G?q/11 and G?i1,2 are activated by pancreastatin receptors in rat liver: Studies with GTP-?35S and azido-GTP-?-32P

In the liver, pancreastatin exerts a glycogenolytic effect through interaction with specific receptors, followed by activation of phospholipase C and guanylate cyclase. Pancreastatin receptor seems to be coupled to two different G protein systems: a pertussis toxin‐insensitive G protein that mediates activation of phospholipase C, and a pertussis toxin sensitive G protein that mediates the cyclic GMP production. The aim of this study was to identify the specific G protein subtypes coupling pancreastatin receptors in rat liver membranes. GTP binding was determined by using γ‐35S‐GTP; specific anti‐G protein α subtype sera were used to block the effect of pancreastatin receptor activation. Activation of G proteins was demonstrated by the incorporation of the photoreactive GTP analogue 8‐azido‐α‐32P‐GTP into liver membranes and into specific immunoprecipitates of different Gα subunits from soluble rat liver membranes. Pancreastatin stimulation of rat liver membranes increases the binding of γ‐35S‐GTP in a time‐ and dose‐dependent manner. Activation of the soluble receptors still led to the pancreastatin dose‐dependent stimulation of γ‐35S‐GTP binding. Besides, WGA semipurified receptors also stimulates GTP binding. The binding was inhibited by treatment with anti‐Gαq/11 (85%) and anti‐Gαi1,2 (15%) sera, whereas anti‐Gαo,i3 serum failed to affect the binding. Finally, pancreastatin stimulates GTP photolabeling of particulate membranes. Moreover, it specifically increased the incorporation of 8‐azido‐α‐32P‐GTP into Gαq/11 and Gα, but not into Gαo,i3 from soluble rat liver membranes. In conclusion, pancreastatin stimulation of rat liver membranes led to the activation of Gαq/11 and Gαi1,2 proteins. These results suggest that Gαq/11 and Gαi1,2 may play a functional role in the signaling of pancreastatin receptor by mediating the production of IP3 and cGMP respectively. J. Cell. Biochem. 73:469–477, 1999.

Pancreastatin, a chromogranin A-derived peptide, activates Gα16 and phospholipase C-β2 by interacting with specific receptors in rat heart membranes

Abstract Pancreastatin (PST) is one of the chromogranin A (CGA)-derived peptides with known biological activity. It has a general inhibitory effect on secretion in many exocrine and endocrine systems including the heart atrium. Besides, a role of PST as a counter-regulatory peptide of insulin action has been proposed in the light of its effects on glucose and lipid metabolism in the liver and adipose tissue, where receptors and signaling have been described. Gα q/11 pathway seems to mediate PST action. Since PST has been shown to function as a typical calcium-dependent hormone, and increased plasma levels have been found in essential hypertension correlating with catecholamines, we sought to study its possible interaction and signaling in heart membranes. Here, we are characterizing specific PST binding sites and signaling in rat heart membranes. We have found that PST receptor has a K d of 0.5 nM and a B max of 34 fmol/mg of protein. The PST binding is inhibited by guanine nucleotides, suggesting the functional coupling of the receptor with GTP binding proteins (G proteins). Moreover, PST dose-dependently increases GTP binding to rat heart membranes. Finally, we have studied PST signaling-effector system by measuring phospholipase C (PLC) activity using blocking antibodies against different G proteins and PLC isoforms. We have found that PST stimulates PLCβ 2 >PLCβ 1 >PLCβ 3 by activating Gα 16 in rat heart membranes. These data suggest that PST may modulate the cardiac function.

Characterization of pancreastatin receptor and signaling in rat HTC hepatoma cells

Pancreastatin, a chromogranin A-derived peptide widely distributed throughout the neuroendocrine system, has a general inhibitory effect on endocrine secretion and a counterregulatory effect on insulin action. We have recently described the cross-talk of pancreastatin with insulin signaling in rat hepatoma cells (HTC), where it inhibits insulin action and signaling through the serine phosphorylation of the insulin receptor, thereby impairing tyrosine kinase activity. Here, we have characterized pancreastatin receptors and signaling in HTC cells. The pancreastatin effector systems were studied by determining phospholipase C activity in HTC membranes and mitogen-activated protein kinase (MAPK) phosphorylation activity in HTC cells. Binding studies with radiolabeled pancreastatin showed a population of high affinity binding sites, with a B(max) of 8 fmol/mg protein and a K(d) of 0.6 nM. Moreover, we assessed the coupling of the receptor with a G protein system by inhibiting the binding with guanine nucleotide and by measuring the GTP binding to HTC membranes. We found that pancreastatin receptor was coupled with a G alpha(q/11) protein which activates phospholipase C-beta(1) and phospholipase C-beta(3), in addition to MAPK via both beta gamma and alpha(q/11).

Pancreastatin activates β3 isoform of phospholipase C via Gα11 protein stimulation in rat liver membranes

Abstract Pancreastatin (PST) receptors have been recently shown to mediate activation of phospholipase C (PLC) in rat liver membranes. There is evidence that the G protein that links pancreastatin receptor with PLC-β is pertussis toxin-insensitive and belongs to the Gαq family. Here, we have employed blocking antisera to sort out the specific PLC-β isoform as well as the specific Gα subunit activated by PST receptor in rat liver membranes. The presence of different PLC-β isoforms was checked by immunoblot analysis. Only PLC-β4 was not detected, whereas PLC-β1, β2 and β3 were abundant in rat liver membranes. However, only anti-PLC-β3 serum was able to block the PST receptor response. We also checked the expression of Gαq and Gα11 in rat liver membranes by immunoblot. Even though both isoforms were present, only anti-Gα11 serum was able to block the PST receptor response. In order to check the specificity of the blocking antisera, we employed them to block the effect of ADP and thrombin stimulating PLC activity in platelet membranes, a system lacking Gα11. Anti-Gαq but not anti-Gα11 sera were able to block the agonist stimulated PLC activity. These data suggest that PST receptor response is mediated by the activation of the β3 isoform of PLC via Gα11 protein stimulation in rat liver membranes.

Pancreastatin Signaling in the Liver

Pancreastatin (PST) is a 49 amino acid peptide with a carboxyl-terminal glycinamide that was first isolated from porcine pancreas (1). It arises from proteolytic cleavage of its precursor chromogranin A (CGA), a glycoprotein present in neuroendocrine cells, including the endocrine pancreas (2, 3). In islets, PST appears to be localized to the insulin-containing β-cells, somatostatin containing δ-cells (4) and glucagon containing α-cells (5, 6). Besides, post-secretory processing of CGA also occurs (7, 8). Rat CGA A cDNA revealed the existence of a pancreastatin-like sequence, homologous to porcine pancreastatin (9-12).

Role of Leptin in the Immune System

Adipose tissue is no longer considered mere energy storage, but an important endocrine organ that produces many signals in a tightly regulated manner. Leptin is one of the most important hormones secreted by the adipocyte, with a variety of physiological roles related with the control of metabolism and energy homeostasis. One of these functions is the connection between nutritional status and immune competence. The adipocyte-derived hormone leptin has been shown to regulate the immune response both in normal as well as in pathological conditions. Leptin ́s modulation of the immune system is exerted at the development, proliferation, anti-apoptotic, maturation, and activation levels. The role of leptin in regulating immune response has been assessed in vitro as well as in clinical studies. Both the innate and adaptative immune responses are regulated by leptin. Every cell type involved in immunity can be modulated by leptin. In fact, leptin receptors have been found in neutrophils, monocytes, and lymphocytes, as well as belonging to the family of class I cytokine receptors. Moreover, leptin activates similar signaling pathways to those engaged by other members of the family. The overall leptin action in the immune system is a proinflammatory effect, activating proinflammatory cells, promoting T-helper 1 responses, and mediating the production of the other proinflammatory cytokines, such as tumor necrosis factor, interleukin (IL)-2, or IL-6. Leptin receptor is also upregulated by proinflammatory signals. It has been shown that conditions of reduced leptin production are associated with increased infection susceptibility. Conversely, immune-mediated disorders such as autoimmune diseases are associated with increased secretion of leptin and production of proinflammatory pathogenic cytokines. Thus, leptin is a mediator of the inflammatory response, and could have also a permissive role in the development of autoimmune diseases.