Pancreastatin receptor is coupled to a guanosine triphosphate-binding protein of the G<sub>g/11</sub>α family in rat liver membranes

2000

Diabetes

Pancreastatin (PST), a chromogranin A-derived peptide, has counterregulatory effects on insulin in the hepatocyte and the adipocyte, suggesting a possible role in insulin resistance. The mechanism of PST action on glucose and lipid metabolism is typical of a calcium-mobilizing hormone and involves a receptor G q /11 protein-phospholipase C (PLC)-␤ pathway. In the rat adipocyte, PST inhibits insulin-mediated glucose transport, glucose utilization, and lipid synthesis, and it has a lipolytic effect but stimulates basal and insulinstimulated protein synthesis. We have also recently studied the PST receptor-effector system in adipocyte membranes. To further investigate the mechanisms of PST effect on insulin action, we studied the cross-talk of PST with insulin signaling in the rat adipocyte. We found that PST inhibits insulin-stimulated GLUT4 translocation to the membrane, which may explain the reported inhibition of glucose transport. Tyrosine phosphorylation of the activated insulin receptor, insulin receptor substrate (IRS)-1, and p60-70 was also blunted, preventing their association with p85 phosphatidylinositol 3-kinase (PI3K) and their activity. The mechanism of this inhibition involves the activation of the "classical" protein kinase C isoforms and the serine phosphorylation of insulin receptor and IRS-1. On the other hand, PST activates the mitogen-activated protein kinase (MAPK) signaling module and enhances the effect of insulin. This pathway may account for the described effect of PST on protein synthesis. In conclusion, PST seems to inhibit the insulin-stimulated PI3K pathway in the adipocyte, whereas it activates the MAPK pathway. These data provide some clues to the PST cross-talk with insulin signaling that may explain the PST effects on glucose metabolism and protein synthesis. Diabetes 49:1288-1294, 2000 P ancreastatin (PST) is a chromogranin A-derived peptide (1,2) widely distributed throughout the neuroendocrine system (3-6). In islets, PST is present in ␤-, ␣-, and ␦-cells (4-6). PST may be secreted from the neuroendocrine cells after the precursor glycoprotein chromogranin A is processed (7,8). Postsecretory processing of chromogranin A also occurs (9,10). A PSTlike sequence has been found in different species, including the rat (11-13).PST was named after its first described effect inhibiting insulin secretion (1). However, many other different biologic effects have been reported (14). The best characterized effect of PST was studied in the rat liver (15), in which it has a glycogenolytic effect (16-18) and a counterregulatory effect to insulin (19). PST action in the liver is mediated by a specific G protein-coupled receptor (20-22), activating G␣ q/11 , which then activates PLC-␤3 in the plasma membrane (23). This signaling pathway leads to an increase in intracellular calcium concentration ([Ca 2+ ] i ) and activation of protein kinase C (PKC), which may finally mediate PST action (24,25). Recently, we found that PST also has metabolic effects in rat adipocytes (26). Thus, PST has a li...

mentioning

confidence: 99%

Pancreastatin modulates insulin signaling in rat adipocytes: mechanisms of cross-talk.

González-Yanes

2000

Diabetes

“…Nonspecific binding was determined in the presence of 10 − 5 M cold GTP-k-S. All assays were performed in quadruplicate. Blocking experiments with antisera were performed by the addition of the specific antisera (1:100 final dilution) to the plasma membranes and incubation for 2 h at 4°C with gentle agitation as previously described [31][32][33]. The 1:100 dilution of sera was found to be the dilution with maximum blocking effect as observed in previous work [31][32][33].…”

mentioning

confidence: 99%

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

CMLS, Cell. Mol. Life Sci.

González-Yanes

Santos‐Álvarez

et al. 1999

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-35S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the G alpha i/G alpha o family. The expression of these G alpha proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific G alpha protein activated by insulin stimulation. Anti-G alpha il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-G alpha o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with G alpha il,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of G alpha il,2 protein by insulin. These findings suggest that G alpha il,2 proteins might be involved in insulin action.

“…We have shown that its action in the liver is mediated by a specific receptor [17,18] coupled to GTP binding proteins in the plasma membrane [17±19]. This receptor activates Ga q/11 in rat liver membranes [20] resulting in activation of Diabetologia (1999) Summary Pancreastatin, a neuropeptide derived from chromogranin A, has a glycogenolytic and counterregulatory effect to insulin in the rat liver. This effect is mediated by calcium and protein kinase C activity.…”

mentioning

confidence: 99%

Modulation of insulin receptor signalling by pancreastatin in HTC hepatoma cells

1999

Diabetologia

Pancreastatin [1], a chromogranin A-derived neuropeptide [2] that was isolated first from the porcine pancreas is widely distributed throughout the neuroendocrine system [3±6]. In islets, it appears to be localized to the insulin-containing beta cells, somatostatin containing delta cells [4] and glucagon containing alpha cells [5,6]. Post-secretory processing of chromogranin A also occurs [7,8]. A pancreastatinlike sequence, homologous to porcine pancreastatin has been found in rat chromogranin A cDNA [9±11].Pancreastatin has a number of effects in a variety of cell targets [12]. Thus, we have found a glycogenolytic effect [13±15] and a counterregulatory effect to insulin in the rat liver [16]. We have shown that its action in the liver is mediated by a specific receptor [17,18] coupled to GTP binding proteins in the plasma membrane [17±19]. This receptor activates Ga q/11 in rat liver membranes [20] resulting in activation of Diabetologia (1999) Summary Pancreastatin, a neuropeptide derived from chromogranin A, has a glycogenolytic and counterregulatory effect to insulin in the rat liver. This effect is mediated by calcium and protein kinase C activity. Our aim was to study the possible cross-talk between pancreastatin and the insulin signalling system, by using the well-studied insulin sensitive rat hepatoma HTC cells. First, we checked the counterregulatory effect of pancreastatin on insulin action. Pancreastatin dose-dependently inhibited insulin stimulated glycogen synthesis. This effect was not due to competition for insulin receptors. Moreover, when protein kinase C activation was blocked with staurosporine, this effect of pancreastatin was not observed. Next, we found a dose-dependent inhibition of insulin receptor autophosphorylation by pancreastatin. In addition, phosphorylation of the major substrates of insulin receptor in HTC, i. e. insulin-receptor substrate (IRS)-1/IRS-2 and p62 was also blunted and so was its association with p85 regulatory subunit of phosphatidylinositol-3-kinase. Moreover, the insulin activation of S6 kinase was also blocked by pancreastatin. Again, all these inhibitory effects of pancreastatin were prevented by staurosporine. Furthermore, pancreastatin produced Ser/Thr phosphorylation of insulin receptor by a staurosporine-sensitive mechanism. Finally, we checked the pancreastatin activation of protein kinase C in HTC cells and found that a ªclas-sicalº isoform of this protein is translocated to the plasma membrane. These findings suggest that pancreastatin could exert its anti-insulin effect in the hepatocyte by interrupting the stimulation of early insulin receptor signalling as a result of phosphorylation. This interaction might have a role in the mechanisms of insulin resistance. [Diabetologia (1999) Corresponding author: V. Sµnchez-Margalet, Department Medical Biochemistry and Molecular Biology, School of Medicine, Av. Sanchez Pizjuan 4, E-41 009 Seville, Spain Abbreviations: IRS-1, Insulin receptor substrate 1; IRS-2, insulin receptor substrate 2; PI3K, phosphatidyli...