Gastric leptin, but not estrogen and somatostatin, contributes to the elevation of ghrelin mRNA expression level in fasted rats

Ghrelin, an endogenous ligand for the GH secretagog receptor, is predominantly produced in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased after refeeding. It has also been reported that estrogen upregulates ghrelin expression and production and that somatostatin inhibits ghrelin secretion, whereas leptin has a paradoxical effect. Recently, several studies have shown that estrogen, somatostatin, and leptin are produced in the stomach, but the direct effects of these gastric hormones on ghrelin expression in a fasting state remain obscure. In this study, we examined the mRNA expression levels of gastric ghrelin, aromatase (estrogen synthetase), leptin and somatostatin, and concentrations of stomach leptin and portal vein 17beta-estradiol in fasted male rats. After 48 h of fasting, although gastric ghrelin mRNA level was significantly increased, both gastric leptin mRNA level and leptin content were decreased. Further, refeeding of fasted rats resulted in a decrease in ghrelin expression level and an increase in leptin expression level. On the other hand, gastric estrogen and somatostatin levels did not change after fasting. In vitro studies revealed that leptin dose-dependently inhibited ghrelin expression and also inhibited estrogen-stimulated ghrelin expression. Moreover, ghrelin cells were found to be tightly surrounded by leptin cells. RT-PCR analysis clearly showed that long and short forms of the leptin receptor are expressed in the rat stomach. These results strongly suggest that an elevated gastric ghrelin expression level in a fasting state is regulated by attenuated restraint from decreased gastric leptin level.     

Leptin but not neuropeptide Y up-regulated glucagon-like peptide 1 receptor expression in GT1-7 cells and rat hypothalamic slices

In an attempt to gain better insight into the central effects of glucagon-like peptide (GLP-1), we studied the action of glucose and of regulatory peptides on the expression of its receptor (GLP-1R) in hypothalamic GT1-7 cells and in ventromedial (VMH) and lateral (LH) rat hypothalamus slices. The promoter activity of GLP-1R in transfected GT1-7 cells increased with leptin, whereas neuropeptide Y (NPY) did not modify it. Interestingly, when cells were incubated with both NPY and leptin, NPY blocked the stimulating effect of leptin. The effects of leptin and NPY were also confirmed at messenger RNA levels. In hypothalamic slices, GLP-1R messenger RNA levels increased at higher glucose concentrations in the VMH. In addition, leptin exerted a stimulating effect; and NPY did not modify receptor expression. By contrast, in the LH, the opposite effects were found for those parameters, except at 20 mmol/L glucose. These findings suggest that the stimulating effect of leptin on GLP-1R expression, with no changes in NPY-induced activity, could enhance the anorexic actions generated through this receptor. In addition, the different responses of the VMH and LH may be related to specific functions of these structures, as already known in vivo, highlighting the interest of hypothalamic slices for this kind of study.     

Stimulation of the gonadotropic axis by the neuropeptide Y receptor Y1 antagonist/Y4 agonist 1229U91 in the male rat

Neuropeptide Y (NPY) is a highly potent orexigenic substance that is also known to modulate gonadotropin secretion. Five receptor subtypes for NPY have been identified, and a potent antagonist for the receptor subtype 1 (Y1), 1229U91, also known as GW1229 or GR231118, has been described. Subsequently, 1229U91 was also shown to represent a highly potent agonist for the Y4 receptor subtype. Very unexpectedly, intracerebroventricular administration of 1229U91 elicited an intense, dose-dependent surge of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in intact male rats that lasted for 6 h. Such stimulation was absent when a potent gonadotropin-releasing hormone antagonist was administered systemically, suggesting that 1229U91 acts centrally to stimulate gonadotropin-releasing hormone release. 1229U91 administration had no effect on growth hormone, thyroid-stimulating hormone, and corticosterone secretions. In addition to 1229U91, four other parent dimer molecules described earlier produced a marked and sustained stimulation of LH when injected intracerebroventricularly that was proportional to their binding affinity for the Y4 receptor. Central administration of the specific Y1 antagonist BIBO3304 (20 microgram) had no effect on LH secretion, making it unlikely for 1229U91 to stimulate LH secretion by an antagonistic action on the Y1 receptor subtype, thus suggesting a Y4 receptor mediation. In conclusion, the 1229U91 molecule displays an interesting conformational epitope that is able to generate large LH surges, possibly by activating Y4 or Y4-like receptor subtypes or by acting on a NPY receptor unrelated target.     

Acute decrease in circulating T3 levels enhances, but does not normalise, the GH response to GHRP-6 plus GHRH in thyrotoxicosis

In thyrotoxicosis there is an impaired GH response to GHRH, normal GH responsiveness to GHRP-6 and lack of synergistic GH response after simultaneous administration of both peptides. We have previously shown that the GHRH-induced GH release in these patients increases after an acute reduction of circulating T3 values with administration of iopanoic acid, a compound that inhibits peripheral conversion of T4 to T3. We have now studied the effect of a decrease in serum T3 levels on the GH response to GHRP-6 (1 microg/kg) plus GHRH (100 microg) in 9 hyperthyroid patients before and after 15 days of treatment with iopanoic acid (3 g every 3 days) and propylthiouracil (600 mg/day). Nine normal subjects were also studied. In all hyperthyroid patients iopanoic acid induced a rapid decrease and normalisation of serum T3 levels. In these subjects peak GH (microg/l; mean +/- SE) and AUC (microg/l x 120 min) values after GHRP-6 plus GHRH were significantly higher on day 15 compared to pretreatment values (peak, 18.3 +/- 3.0 vs 13.4 +/- 1.9; AUC, 1227.9 +/- 212.9 vs 968.5 +/- 160.4; p<0.05). Despite the significant enhancement of the GH responsiveness to GHRP-6 plus GHRH after treatment with iopanoic acid, this response remained significantly blunted when compared to controls both in terms of peak GH (18.3 +/- 3.0 vs 83.7 +/- 15.2; p<0.05) and AUC values (1227.9 +/- 212.9 vs 4956.5 +/- 889.3; p<0.05). In conclusion, our results show that an acute decrease of circulating T3 levels enhances, but does not normalise, the GH response to GHRP-6 plus GHRH in thyrotoxicosis. This could suggest that circulating T3 does not have a major role in the mechanisms involved in the synergistic effect of these peptides.     

Protease‐activated receptor 2, rather than protease‐activated receptor 1, contributes to the aggressive properties of synovial fibroblasts in rheumatoid arthritis

Objective

To investigate whether protease‐activated receptor 1 (PAR‐1) and/or PAR‐2 promotes the invasiveness/proliferation of synovial fibroblasts (SFs) and to determine the signaling mechanisms of these pathways.

Methods

SFs were isolated from the synovial tissue of patients with rheumatoid arthritis (RA), patients with osteoarthritis (OA), and PAR‐1– or PAR‐2–knockout (KO) mice. Expression of PAR‐1 and PAR‐2 was detected by immunofluorescence and Western blotting. The invasion and proliferation of SFs were measured by invasion assay and MTT assay, respectively. Matrix metalloproteinase 2 (MMP‐2) and MMP‐9 were detected by zymography, and cytokines were measured by enzyme‐linked immunosorbent assay.

Results

PAR‐1 and PAR‐2 were colocalized with SFs in RA and OA synovium and, to a considerably lesser extent, in normal synovium. Inhibition of PAR‐2 by small interfering RNA (siRNA) inhibited RASF invasion and proliferation, whereas blocking of PAR‐1 by siRNA had the reverse effects. SFs from PAR‐2–KO mice exhibited slower rates of proliferation and invasion. SFs from PAR‐1–KO mice produced less MMP‐2 and, in response to tumor necrosis factor α (TNFα) stimulation, had increased MMP‐9 secretion when compared to SFs from wild‐type and PAR‐2–KO mice. Inhibition of PAR‐1, but not PAR‐2, stimulated the secretion of interleukin‐17 (IL‐17) and TNFα by RASFs. Furthermore, PAR‐1 and PAR‐2 had opposing effects on the activation of ERK, p38, and NF‐κB.

Conclusion

Activation of PAR‐1 stimulates MMP‐2 secretion, inhibits RASF growth and invasion, and decreases production of IL‐17 and TNFα by RASFs, whereas activation of PAR‐2 stimulates RASF growth and invasion and increases production of TNFα. Thus, although PAR‐1 and PAR‐2 are coexpressed by RASFs, PAR‐2 alone appears to be responsible for the aggressive properties of RASFs and is likely to contribute to the pathologic progression of RA.

     

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin,but neither is required for regulation of phospholipase C

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.     

Expression of an activated erythropoietin or a colony-stimulating factor 1 receptor by pluripotent progenitors enhances colony formation but does not induce differentiation.

Whether the presence of specific receptors on the surface of developing cells is the cause or consequence of lineage restriction is not known. If activation of specific receptors is the driving event in differentiation, the premature expression of specific receptors would promote differentiation along that pathway. In this study pluripotent progenitors, obtained from blast cell colonies (pooled or individual) of 5-fluorouracil-treated mice, were infected with retroviral vectors containing either an activated receptor for erythropoietin (EPO), an erythroid progenitor growth factor, or the receptor for colony-stimulating factor 1 (CSF-1), a macrophage growth factor. These receptors exhibit expression patterns restricted to committed progenitors. The developmental potential of infected pluripotent progenitors was not changed, although they expressed the exogenous genes, suggesting that in these cells activation of lineage-specific receptors does not induce differentiation. Acquisition of a constitutively activated EPO receptor allowed erythroid development in mixed colonies in the absence of EPO, as expected. Infection of progenitors with a virus containing the CSF-1 receptor promoted the development of granulocyte/macrophage (GM) colonies but did not alter the differentiation potential of either colony-forming unit (CFU)-GM or CFU-mix.     

Adipose tissue loss in adjuvant arthritis is associated with a decrease in lipogenesis, but not with an increase in lipolysis

Adjuvant-induced arthritis is a model of rheumatoid arthritis that induces cachexia. In other cachectic situations, there is an increase in lipolysis resulting in a loss of adipose tissue mass. The aim of this work was to analyse the effect of chronic arthritis, induced by adjuvant injection, on white adipose tissue (WAT). For this purpose, rats were killed 10 days after adjuvant injection, when the first external symptoms appeared, on days 15 and 22 when the external signs of the illness reach their severest level. As arthritis decreases food intake, a pair-fed group was also included. Serum concentrations of insulin, leptin, adiponectin, glycerol and nitrites, as well as gene expression of leptin, adiponectin, hormone-sensitive lipase (HSL), fatty acid synthase (FAS), tumour necrosis factor alpha and zinc-alpha(2)-glycoprotein (ZAG) were determined. Arthritis decreased food intake between days 5 and 16, but not during the last 5 days of the experiment. There was a marked decrease in relative adipose tissue weight and in serum leptin and adiponectin as well as in their gene expression in WAT in arthritic rats. Arthritis decreased the gene expression of FAS in the WAT. However, none of these effects was found in pair-fed rats. Arthritis did not increase lipolysis, since arthritic rats have lower serum concentrations of glycerol, HSL mRNA in WAT, as well as liver ZAG mRNA than the pair-fed or control rats. These data suggest that in chronic arthritis the decrease in white adipose mass is secondary to a reduced adipose lipogenesis, and this effect is not mainly due to the decrease in food intake.     

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein’s ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure–function relationships of the YchF subfamily of G proteins in all kingdoms of life.GTP-binding proteins (G proteins) play important roles in diverse fundamental biological processes in living organisms, including signal transduction, cell division, development, intracellular transport, translation, and others (1, 2). The functions and working mechanisms of some ancestral G proteins, which are believed to play essential roles in cellular processes, are still unknown. One such group is the Obg family, which features an N-terminal glycine-rich sequence, followed by a G domain for nucleotide binding and a C-terminal TGS (ThrRS, GTPase, and SpoT) domain that has nucleic acid binding affinity (3).A unique subgroup within the Obg family, the YchF proteins, exhibit relaxed nucleotide-binding specificities (4–6). All G proteins contain a G domain (composed of the G1–G5 motifs) for GTP binding and hydrolysis (4). The G4 motif (canonical sequence: NKxD) confers the specificity for binding GTP (4). In YchFs, the noncanonical G4 sequence (NxxE) is correlated with the loss of nucleotide-binding specificities (4, 5). In some cases, ATP is the preferred ligand (4, 5). However, the physiological significance of the ancient and evolutionarily conserved YchF proteins having the relaxed binding specificities for both ATP and GTP is still unknown.Only a few reports touch on the biological functions of YchF proteins and most focus on human and microorganisms (7–9). The YchF proteins may play a role in iron utilization and regulation of the Ton system in Brucella melitensis (7), and in the growth of the procyclic forms of Trypanosoma cruzi (5). The human YchF homolog hOLA1 suppresses cellular oxidative responses (9) but enhances heat shock responses (10). In plants, we have shown that rice (OsYchF1) and Arabidopsis (AtYchF1) YchF proteins function as negative regulators in both abiotic stress responses (11) and plant-defense responses (12).The structures of bacterial, yeast, and human YchF proteins were reported (4, 6). In this study, we have successfully resolved the first crystal structure of a plant YchF homolog (OsYchF1) and, in addition, its cocrystal structures with AMPPNP (adenylyl-imidodiphosphate) and GMPPNP (guanylyl-imidodiphosphate). Our structures explain the dual specificity of OsYchF1 for both ATP and GTP. Using site-directed mutagenesis, we converted the noncanonical G4 motif of OsYchF1 to the canonical sequence of most regular G proteins to test the effect of losing the ATP-binding ability, while retaining GTP binding, on its physiological functions in both plant-defense responses and abiotic-stress responses.     

Aerobic exercise training increases circulating insulin-like growth factor binding protein-1 concentration, but does not attenuate the reduction in circulating insulin-like growth factor binding protein-1 after a high-fat meal

Insulin-like growth factor binding protein-1 (IGFBP-1) has metabolic effects throughout the body, and its expression is regulated in part by insulin. Circulating IGFBP-1 predicts development of cardiometabolic diseases in longitudinal studies, and low IGFBP-1 concentrations are associated with insulin resistance and consumption of a high-fat diet. Because of the favorable metabolic effects of regular aerobic exercise, we hypothesized that aerobic exercise training would increase plasma IGFBP-1 concentrations and attenuate the reduction in IGFBP-1 after a high-fat meal. Ten overweight (body mass index = 28.7 ± 0.9 kg/m(2)), older (61 ± 2 years) men and women underwent high-fat feeding and oral glucose tolerance tests at baseline and after 6 months of aerobic exercise training. In response to aerobic exercise training, subjects increased cardiorespiratory fitness by 13% (P < .05) and insulin sensitivity index by 28% (P < .05). Basal plasma concentrations of IGFBP-1 increased by 41% after aerobic exercise training (P < .05). The insulin response to an oral glucose tolerance test was a significant predictor of fasting plasma IGFBP-1 concentrations at baseline and after exercise training (P = .02). In response to the high-fat meal at baseline, plasma IGFBP-1 concentrations decreased by 58% (P < .001); a 61% decrease to similar postprandial concentrations was observed after exercise training (P < .001). Plasma insulin response to the high-fat meal was inversely associated with postprandial IGFBP-1 concentrations at baseline and after exercise training (P = .06 and P < .05, respectively). Although aerobic exercise training did not attenuate the response to a high-fat meal, the increase in IGFBP-1 concentrations after exercise training may be one mechanism by which exercise reduces risk for cardiometabolic diseases in older adults.     

Circulating ghrelin levels in newborns are not associated to gender,body weight and hormonal parameters but depend on the type of delivery

Ghrelin, a new gastric-derived hormone, probably plays a major role in managing energy balance and the neuroendocrine response to starvation. Information about the age-related variation in ghrelin secretion is scanty. We measured circulating ghrelin levels in 93 full term newborns adequate for gestational age, in 39 normal children and in 19 lean healthy adults. Our findings demonstrate that ghrelin levels are independent of age and gender from birth to adulthood. Interestingly, ghrelin secretion at birth is not associated to body weight and hormonal parameters such as GH, insulin and leptin levels. On the other hand, ghrelin levels seem dependent on the type of delivery, being lower in newborns after caesarean section with respect to those after normal delivery.     

Endogenous interleukin-6, but not tumor necrosis factor alpha, contributes to the development of toll-like receptor 4/myeloid differentiation factor 88-mediated acute arthritis in mice

OBJECTIVE: To generate a mouse model of reactive arthritis (ReA), an aseptic synovitis that develops in joints distant from the primary bacterial infection site, to examine roles for Toll-like receptors (TLRs) that recognize bacterial components involved in the development of this arthritis, and to identify the cytokine(s) relevant to this arthritis. METHODS: Mice were treated with cell wall extract from Escherichia coli (ECW) gram-negative bacterium by injection into the footpads. Seven days later, the mice were challenged with lipopolysaccharide (LPS), a TLR-4 ligand, which was injected into the knee joint cavity. To investigate the cytokine(s) involved in this arthritis, mice deficient in various arthritogenic cytokines, such as interleukin-6 (IL-6), IL-12, IL-18, interferon-gamma, and tumor necrosis factor alpha (TNFalpha), were sequentially treated with ECW and LPS. RESULTS: ECW-primed mice manifested acute severe arthritis after intraarticular challenge with ECW or LPS, while unprimed mice exhibited modest changes after these challenges. Mutant mice lacking functional TLR-4 or myeloid differentiation factor 88 (MyD88), an adaptor molecule of TLR-4 signaling, were resistant to this arthritis. Although both TNFalpha and IL-6 were equally expressed in the joint after LPS challenge, Il6(-/-) mice, but not Tnf(-/-) mice, were resistant to ECW/LPS-induced arthritis. CONCLUSION: Our present results clearly indicate the importance of priming with ECW and the requirement of TLR-4/MyD88-mediated IL-6, but not TNFalpha, for the development of ECW/LPS-induced arthritis. LPS-induced IL-6, in the absence of TNFalpha, mediates LPS-induced arthritis. These results suggest that IL-6 is a rational target for therapeutic regimens for inflammatory arthritis, including ReA and rheumatoid arthritis.     

Elevated leptin levels are associated with excess gains in fat mass in girls, but not boys, with type 1 diabetes: longitudinal study during adolescence

Adolescents, in particular girls, with type 1 diabetes may gain excessive weight during puberty. We present the results of a longitudinal study aimed to determine the roles of leptin and insulin in changes in body composition in subjects with type 1 diabetes and controls. Forty-six children (23 boys) with type 1 diabetes and 40 controls (20 boys) were followed from 8-17 yr of age. Height, weight, and sc skinfolds were assessed every 6 months, and a blood sample taken for leptin determination. Throughout the age range, body mass index (mean +/- SEM) was greater by 1.45 +/- 0.69 kg/m(2) in girls and 1.46 +/- 0.55 kg/m(2) in boys with type 1 diabetes compared with control values. In girls with type 1 diabetes, this reflected greater percent body fat (3.2 +/- 1.0%; P = 0.002), whereas in boys it related to differences in fat-free mass. Both boys and girls with type 1 diabetes had higher leptin levels adjusted for percent body fat than controls; in the girls this was related to insulin dose (regression coefficient B = 0.006 +/- 0.003; P = 0.04) and greater gains in fat mass. Hyperinsulinemia and raised leptin levels are associated with gains in fat mass throughout puberty in girls, but not boys, with type 1 diabetes.     

Epigenetic dysregulation of the Jak/STAT pathway by frequent aberrant methylation of SHP1 but not SOCS1 in acute leukaemias

SOCS1 and SHP1 are negative regulators of the Jak/STAT signalling pathway that is implicated in leukaemogenesis. We studied if aberrant methylation of SOCS1 and SHP1 might be involved in the pathogenesis and prognostication of acute leukaemias by methylation-specific polymerase chain reaction (MSP). At diagnosis, methylation of SHP1 occurred more frequently in acute myeloid leukaemia (AML) (n=26, 52%) than acute lymphoblastic leukaemia (ALL) (n=6, 24%) (p=0.02). Methylation of SOCS1 was absent in both AML and ALL patients. SHP1 methylation was not associated with specific clinicopathologic features and had no prognostic impact on AML patients. Frequent methylation of SHP1, but not SOCS1, may be important in the pathogenesis, but not prognosis, of acute leukaemias.     

Adiponectin, leptin, and erythrocyte sodium/lithium countertransport activity, but not resistin, are related to glucose metabolism in growth hormone-deficient adults

In a randomized, placebo-controlled, crossover study under metabolic ward conditions, 10 GH-deficient adults received 1-wk GH replacement therapy (9.5 microg/kg.d). The effect of this treatment on the erythrocyte sodium/lithium countertransport (SLC) activity and on serum levels of adiponectin, resistin, leptin, IGF binding protein-1 (IGFBP-1) and IL-6 was determined. The 1-wk GH replacement impaired glucose homeostasis determined from an oral glucose tolerance test. The other measured variables in serum were unchanged by GH replacement. At baseline, serum adiponectin level was inversely correlated and serum leptin level was positively correlated with measures of glucose tolerance and insulin sensitivity. The changes in serum leptin level and erythrocyte SLC activity were positively correlated, and the change in serum IGFBP-1 level was negatively correlated, correlated with changes in measures of glucose metabolism. In conclusion, short-term GH treatment induced glucose intolerance but did not significantly change the erythrocyte SLC activity and the serum levels of adipokines, arguing against direct effects of GH on these measures. However, baseline values or changes in erythrocyte SLC activity, adiponectin, leptin, and IGFBP-1 correlated with glucose metabolism. This suggests that these factors are of importance for glucose homeostasis in GH-deficient adults, most likely through GH-independent mechanisms.