Src kinases mediate VEGFR2 transactivation by the osteostatin domain of PTHrP to modulate osteoblastic function.

Parathyroid hormone-related protein (PTHrP) stimulates osteoblastic function through its N- and C-terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107-139) and the shorter PTHrP (107-111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3-E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr-1059 within 5-10 min in both MC3T3-E1 and rat osteoblastic osteosarcoma UMR-106 cells. This phosphorylation elicited by osteostatin appears to be VEGF-independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal-regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin-dependent induction of ERK and Akt activation was abrogated by SU6656. Up-regulation of VEGF and osteoprotegerin gene expression as well as the pro-survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C-terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function.

Characterization of skeletal alterations in a model of prematurely aging mice.

An age-related bone loss occurs, apparently associated with the concomitant increase in an oxidative stress situation. However, the underlying mechanisms of age-related osteopenia are ill defined since these studies are time consuming and require the use of many animals (mainly rodents). Here, we aimed to characterize for the first time the bone status of prematurely aging mice (PAM), which exhibit an increased oxidative stress. Tibiae from adult (6 months) PAM show an increase in bone mineral density (BMD) and bone mineral content (assessed by bone densitometry) versus those in their normal counterparts (non-prematurely aging mice, NPAM) and similarly decreased in both kinds of mouse with age. However, at this bone site, trabecular BMD (determined by µ-computerized tomography) was similar in both adult PAM and old (18 months) NPAM. Femurs from these groups of mice present an increase in oxidative stress, inflammation, osteoclastogenic, and adipogenic markers, but a decrease in the gene expression of osteoblastic differentiation markers and of the Wnt/ß-catenin pathway. Our findings show that adult PAM recapitulate various age-related bone features, and thus are a suitable model for premature bone senescence studies.

Normalizing action of exendin-4 and GLP-1 in the glucose metabolism of extrapancreatic tissues in insulin-resistant and type 2 diabetic states.

Exendin-4 (Ex-4) mimics glucagon-like peptide-1 (GLP-1 or GCG as listed in the HUGO database), being anti-diabetic and anorectic, in stimulating glucose and lipid metabolism in extrapancreatic tissues. We studied the characteristics of Ex-4 and GLP-1 action, during prolonged treatment, on GLUTs expression (mRNA and protein), glycogen content (GC), glucose transport (GT), glycogen synthase a (GSa), and kinase (PI3K and MAPKs) activity, in liver, muscle, and fat of insulin-resistant (IR, by fructose) and type 2 diabetic (T2D, streptozotocin at birth) rats compared with normal rats. In both IR and T2D, the three tissues studied presented alterations in all measured parameters. In liver, GLP-1 and also Ex-4 normalized the lower than normal Glut2 (Slc2a2) expression and showed a trend to normalize the reduced GC in IR, and GLP-1, like Ex-4, also in T2D, effects mediated by PI3K and MAPKs. In skeletal muscle, neither GLP-1 nor Ex-4 modified Glut4 (Slc2a4) expression in either experimental model but showed normalization of reduced GT and GSa, in parallel with the normalization of reduced PI3K activity in T2D and MAPKs in both models. In adipose tissue, the altered GLUT4 expression in IR and T2D, along with reduced GT in IR and increased GT in T2D, and with hyperactivated PI3K in both, became normal after GLP-1 and Ex-4 treatment; yet, MAPKs, that were also higher, became normal only after Ex-4 treatment. The data shows that Ex-4, as well as GLP-1, exerts a normalizing effect on IR and T2D states through a distinct post-receptor mechanism, the liver being the main target for Ex-4 and GLP-1 to control glucose homeostasis.

Characteristic of GLP-1 effects on glucose metabolism in human skeletal muscle from obese patients.

Direct effects of GLP-1, kinase-mediated, on glucose and lipid metabolism in rat and human extrapancreatic tissues, are amply documented and also changes in type-2 diabetic (T2D) patients. Here, we explored the characteristics of the GLP-1 action and those of its analogs Ex-4 and Ex-9, on muscle glucose transport (GT) and metabolism in human morbid obesity (OB), as compared with normal and T2D subjects. In primary cultured myocytes from OB, GT and glycogen synthase a (GSa) activity values were lower than normal, and comparable to those reported in T2D patients; GT was increased by either GLP-1 or Ex-9 in a more efficient manner than in normal or T2D, up to normal levels; the Ex-4 increasing effect on GSa activity was two times that in normal cells, while Ex-9 failed to modify the enzyme activity. In OB, the control value of all kinases analyzed - PI3K, PKB, MAPKs, and p70s6K - although lower than that in normal or T2D subjects, the cells maintained their response capability to GLP-1, Ex-4, Ex-9 and insulin, with some exceptions. GLP-1 and exendins showed a direct normalizing action in the altered glucose uptake and metabolism in the muscle of obese subjects, which in the case of GLP-1 could account, at least in part, for the reported restoration of the metabolic conditions of these patients after restrictive surgery.

Presence of a functional receptor for GLP-1 in osteoblastic cells, independent of the cAMP-linked GLP-1 receptor.

Glucagon-like peptide 1 (GLP-1) controls glucose metabolism in extrapancreatic tissues through receptors other than the pancreatic cAMP-linked GLP-1 receptor; also, GLP-1 induces an insulin- and PTH-independent bone anabolic action in insulin-resistant and type-2 diabetic rats. Here we searched for the presence and characteristics of GLP-1 receptors in osteoblastic MC3T3-E1 cells. [(125)I]-GLP-1 specific binding to MC3T3-E1 cells was time- and temperature-dependent, reaching maximal value at 30 min at 25 degrees C; in these conditions, [(125)I]-GLP-1 binding was dissociable, and displaced by GLP-1, partially by GLP-2, but not by exendin-4 (Ex-4), exendin-9 (Ex-9), glucagon or insulin; Scatchard analysis of the unlabeled GLP-1 data showed high and low affinity binding sites; cross-linking of GLP-1 binding revealed an estimated 70 kDa band, almost undetectable in the presence of 10(-6) M GLP-1. GLP-1, Ex-9, insulin or glucagon failed to modify cellular cAMP content, while GLP-2 and Ex-4 increased it. However, GLP-1 induced an immediate hydrolysis of glycosylphosphatidylinositols (GPIs) generating short-lived inositolphosphoglycans (IPGs), and an increase in phosphatidylinositol-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) activities; Ex-4 also affected GPIs, but its action was delayed with respect to that of GLP-1. This incretin was found to decrease Runx2 but increased osteocalcin gene expression, without affecting that of osteoprotegerin or the canonical Wnt pathway activity in MC3T3-E1 cells which do not express the pancreatic GLP-1 receptor. Our data demonstrate for the first time that GLP-1 can directly and functionally interact with osteoblastic cells, possibly through a GPI/IPG-coupled receptor.

Characteristics of GLP-1 and exendins action upon glucose transport and metabolism in type 2 diabetic rat skeletal muscle.

Exendin-4, a peptide 53% structurally homologous with glucagon-like peptide 1 (GLP-1), is insulinotropic and has an antidiabetic effect even more prolonged than that of GLP-1. Exendin-9 is an antagonist of GLP-1 receptor and action in several cell systems, but shows GLP-1- and exendin-4-agonistic characteristics in human muscle cells and tissue. The action of GLP-1 upon glucose transport and metabolism in muscle is mediated by specific receptors. In this study we investigated the effect of both exendin-4 and -9, relative to that of GLP-1, upon glucose transport and metabolism in the skeletal muscle from a streptozotocin-induced type 2 diabetic rat model, compared to normal. In normal rats, exendin-4, like GLP-1 and insulin, enhanced glucose uptake. This effect, which is mediated to a certain extent by some kinases (PI3K/ PKB, p70s6k and MAPKs), may be caused by the peptide acting, at least in part, through the muscle GLP-1 receptors. Exendin-9 also stimulated the same kinases, except for PKB, but failed to modify basal glucose uptake. Type 2 diabetic rats showed lower than normal basal muscle glucose transport and oxidation value, and higher glycogen synthase alpha activity and pyruvate release; however, no modification of glucose uptake by GLP-1 or exendin-4 was detected, at variance with insulin, and basal activity of PI3K/PKB was lower than normal, while that of p70s6k and MAPKs was higher. GLP-1 failed to affect the activity of any of the kinases, while exendin-4 increased the activity of PI3K, p70s6k and MAPKs, but not PKB, suggesting that this enzyme plays a major role in exendin-4 effect upon glucose transport in muscle.

Effect of GLP-1 on D-glucose transport, lipolysis and lipogenesis in adipocytes of obese subjects.

GLP-1 has anorectic properties and regulates fuel homeostasis through both its insulinotropic and insulinotrophic actions and effects in extrapancreatic tissue. This study is aimed at characterizing the response to GLP-1 of adipocytes from obese patients, in terms of D-glucose transport and lipid metabolism, in comparison with data from normal subjects. Adipocytes were obtained by enzymatic digestion from the abdominal fat tissue of 25 morbidly obese patients and 8 normal subjects undergoing bariatric or inguinal hernia surgery, respectively. Basal GLUT4 expression, D-glucose transport, glycerol release and lipogenesis were measured in cells treated, when required, with 10(-12)-10(-9) M GLP-1, insulin, glucagon and the GLP-1 structurally related peptides, exendin-4 and exendin-9. In obese patients, versus normal subjects, a trend towards lower values was found in GLUT4 protein or mRNA, although the differences were not statistically significant; insulin-stimulated glucose uptake was higher and cells did not respond to GLP-1, while both exendins (10(-10) and 10(-9) M) exerted an inhibitory action; basal lipolysis was higher and so was the effect of GLP-1 and glucagon, whereas insulin abolished the lipolytic action of all peptides; both basal lipogenesis and the response to insulin were higher while GLP-1 and exendin-4 were ineffective. These results document the analogies and dissimilarities between the response to GLP-1, exendin-4 and exendin-9, as well as to insulin and glucagon, relative to glucose transport and lipid metabolism of fat tissue from obese patients versus normal subjects, the reduced lipogenic effect and enhanced lipolytic action of GLP-1 being, perhaps, adequate for its therapeutic use in obesity.

Effect of GLP-1 on glucose transport and its cell signalling in human myocytes.

Glucagon-like peptide-1 (GLP-1) controls glucose metabolism in extrapancreatic tissues participating in glucose homeostasis, through receptors not associated to cAMP. In rat hepatocytes, activation of PI3K/PKB, PKC and PP-1 mediates the GLP-1-induced stimulation of glycogen synthase. We have investigated the effect of GLP-1 in normal human myocytes, and that of its structurally related peptides exendin-4 (Ex-4) and its truncated form 9-39 (Ex-9) upon glucose uptake, and the participation of cellular enzymes proposed to mediate insulin actions. GLP-1 and both exendins activated, like insulin, PI3K/PKB and p42/44 MAPK enzymes, but p70s6k was activated only by GLP-1 and insulin. GLP-1, Ex-4 and Ex-9, like insulin, stimulated glucose uptake; wortmannin blocked the action of GLP-1, insulin and Ex-9, and reduced that of Ex-4; PD98059 abolished the effect of all peptides/hormones, while rapamycin blocked that of insulin and partially prevented that of GLP-1. H-7 abolished the action of GLP-1, insulin and Ex-4, while Ro 31-8220 prevented only the Ex-4 and Ex-9 effect. In conclusion, GLP-1, like insulin, stimulates glucose uptake, and this involves activation of PI3K/PKB, p44/42 MAPKs, partially p70s6k, and possibly PKC; Ex-4 and Ex-9 both have GLP-1-like effect upon glucose transport, in which both share with GLP-1 an activation of PI3K/PKB--partially in the case of Ex-4--and p44/42 MAPKs but not p70s6k.

Bioactive GLP-1 in gut, receptor expression in pancreas, and insulin response to GLP-1 in diabetes-prone rats.

Glucagon-like peptide-1 (GLP-1) is the most insulinogenic of the glucagon-like peptides secreted mainly by L cells in the small and large intestine in response to the ingestion of nutrients. It binds to a specific GLP-1 receptor (GLP-1R) on beta-cells and can increase islet neogenesis and beta-cell mass. It is not clear whether the transmission of information from the gut to islet beta-cells by messengers such as GLP-1 is different in individuals who develop autoimmune diabetes. In the present study the expression of bioactive GLP-1 protein in the gut and its receptor in the pancreas was examined in diabetes-prone BioBreeding (BBdp) rats in the period before overt diabetes and in age-matched control, non-diabetes-prone BB (BBc) rats. An N-terminal directed antibody specific for the bioactive forms of GLP-1 (GLP-1(7-37) and GLP-1(7-36amide)) was used to mea-sure GLP-1 by radioimmunoassay in proximal, median, and distal gut. Pancreas GLP-1R area fraction, GLP-1R gene expression, and insulin content were analyzed, as were plasma GLP-1, glucose, and insulin. The concentration of GLP-1 protein in the jejunum and ileum of BBdp rats was lower than in BBc rats. Although these animals maintained normal blood glucose, there was impaired pancreatic endocrine function, characterized by low baseline insulin concentration in plasma and pancreas. GLP-1R mRNA expression was threefold less in islets isolated from BBdp rats, and GLP-1R+ islet area fraction in pancreas sections was decreased. When injected iv with GLP-1, BBdp rats displayed lower second-phase insulin response (and insulin/glucose ratios) compared with BBc rats. Thus, young BBdp rats displayed decreased concentrations of bioactive GLP-1 in jejunum and ileum, reduced GLP-1R in islets, and lower second-phase insulin response to iv GLP-1 than controls. The decrease in insulinogenic and islet beta-cell mass-promoting signal from GLP-1 in BBdp rats may contribute to impaired glucoregulation and ineffective maintenance of normal islet mass that shifts islet homeostasis in favor of development of diabetes.