Abdominal surgery inhibits circulating acyl ghrelin and ghrelin-O-acyltransferase levels in rats: role of the somatostatin receptor subtype 2

Clinical studies are evaluating the efficacy of synthetic ghrelin agonists in postoperative ileus management. However, the control of ghrelin secretion under conditions of postoperative gastric ileus is largely unknown. Peripheral somatostatin inhibits ghrelin secretion in animals and humans. We investigated the time course of ghrelin changes postsurgery in fasted rats and whether somatostatin receptor subtype 2 (sst(2)) signaling is involved. Abdominal surgery (laparotomy and 1-min cecal palpation) induced a rapid and long-lasting decrease in plasma acyl ghrelin levels as shown by the 64, 67, and 59% reduction at 0.5, 2, and 5 h postsurgery, respectively, compared with sham (anesthesia alone for 10 min, P < 0.05). Levels were partly recovered at 7 h and fully restored at 24 h. The percentage of acyl ghrelin reduction was significantly higher than that of desacyl ghrelin at 2 h postsurgery and not at any other time point. This was associated with a 48 and 23% decrease in gastric and plasma ghrelin-O-acyltransferase protein concentrations, respectively (P < 0.001). Ghrelin-positive cells in the oxyntic mucosa expressed sst(2a) receptor and the sst(2) agonist S-346-011 inhibited fasting acyl ghrelin levels by 64 and 77% at 0.5 and 2 h, respectively. The sst(2) antagonist S-406-028 prevented the abdominal surgery-induced decreased circulating acyl ghrelin but not the delayed gastric emptying assessed 0.5 h postinjection. These data show that activation of sst(2) receptor located on gastric X/A-like cells plays a key role in the rapid inhibition of circulating acyl ghrelin induced by abdominal surgery while not being primarily involved in the early phase of postoperative gastric ileus.     

Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes

Human saliva, which contains nitrite, is normally mixed with gastric juice, which contains ascorbic acid (AA). When saliva was mixed with an acidic buffer in the presence of 0.1 mM AA, rapid nitric oxide formation and oxygen uptake were observed. The oxygen uptake was due to the oxidation of nitric oxide, which was formed by AA-dependent reduction of nitrite under acidic conditions, by molecular oxygen. A salivary component SCN⁻ enhanced the nitric oxide formation and oxygen uptake by the AA/nitrite system. The oxygen uptake by the AA/nitrite/SCN⁻ system was also observed in an acidic buffer solution. These results suggest that oxygen is normally taken up in the stomach when saliva and gastric juice are mixed.     

Stress-related alterations of acyl and desacyl ghrelin circulating levels: mechanisms and functional implications

Ghrelin is the only known peripherally produced and centrally acting peptide hormone that stimulates food intake and digestive functions. Ghrelin circulates as acylated and desacylated forms and recently the acylating enzyme, ghrelin-O-acyltransferase (GOAT) and the de-acylating enzyme, thioesterase 1/lysophospholipase 1 have been identified adding new layers of complexity to the regulation of ghrelin. Stress is known to alter gastrointestinal motility and food intake and was recently shown to modify circulating ghrelin and GOAT levels with differential responses related to the type of stressors including a reduction induced by physical stressors (abdominal surgery and immunological/endotoxin injection, exercise) and elevation by metabolic (cold exposure, acute fasting and caloric restriction) and psychological stressors. However, the pathways underlying the alterations of ghrelin under these various stress conditions are still largely to be defined and may relate to stress-associated autonomic changes. There is evidence that alterations of circulating ghrelin may contribute to the neuroendocrine and behavioral responses along with sustaining the energetic requirement needed upon repeated exposure to stressors. A better understanding of these mechanisms will allow targeting components of ghrelin signaling that may improve food intake and gastric motility alterations induced by stress.     

Effect of chronic stress on gastric emptying and plasma ghrelin levels in rats

Chronic stress is associated with gastrointestinal functional diseases. Although the pathophysiology seems to be associated with gastrointestinal motility, their mechanisms remain unclear. We investigated gastric emptying and chemical mediators under conditions of continuous stress, which were produced using 8-week-old male Wistar rats kept in a cage filled with water to 2 cm height for 5 days. We examined gastric emptying by the phenol red method and chemical mediators at 4, 8, and 24 h and 3 and 5 days after initiation of stress restraint. Plasma ACTH level was significantly higher in the stress throughout the period of measurement. Continuous stress delayed gastric emptying until 24 h: peak delay was observed at 8 h, whereas gastric emptying was accelerated on days 3 and 5. Plasma noradrenalin level was significantly elevated at every time point until 24 h. Guanethidine pretreatment eliminated the delay in gastric emptying at 8 h. Active ghrelin was significantly increased on days 3 and 5 after peak (at 24 h) plasma total and desacyl ghrelin in the stress group. Number of ghrelin-immunoreactive cells and level of preproghrelin mRNA expression in the gastric body increased in parallel with plasma active ghrelin level. Pretreatment with growth hormone secretagogue receptor antagonist at 5 days partially inhibited the stress-induced acceleration of gastric emptying. Delayed gastric emptying at acute phase of continuous stress was mediated via sympathetic pathway, while acceleration at chronic phase was mediated via increased active ghrelin release from the stomach.     

The effect of high-fat diet on plasma ghrelin and leptin levels in rats

Ghrelin and leptin regulate appetite and energy homeostasis in humans and rodents. The effects of different nutritional factors on ghrelin and leptin secretion are not well documented in rats. Therefore, the aim of our study was to investigate the effect of a high-fat diet on plasma ghrelin and leptin levels and on adiposity. Twenty male Wistar rats, body weight220–260 g, were used in the study. Rats were randomized either on a standard chow diet (n=10) or on a high-fat diet (a mixture of nuts) forad libitum 11-week period. Body weight was measured once per week. At the end of the nutritional period, rats were sacrificed. Blood was collected for determination of lipids and glucose, as well as plasma ghrelin and leptin levels by ELISA method. The weight of different organs was determined. Rats fed on a high-fat diet showed significant increase in total body weight compared to control group. The long-term intake of high-fat diet caused hyperleptinemia and hypoghrelinemia. There was a significant positive correlation between plasma leptin levels and epididymal fat mass, liver and heart. In contrast, ghrelin levels showed inverse correlation with epididymal fat mass and liver weight. In conclusion, long-term intake of high-fat diet induced changes in plasma ghrelin and leptin in male rats, as well as in epididymal fat mass, liver and heart weights.     

Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 μg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 μg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12 h. Ghrelin and desacyl ghrelin (64 μg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.     

Increased expression of TNF-alpha,IL-6, and IL-8 in HALS: implications for reduced adiponectin expression and plasma levels

Human immunodeficiency virus (HIV)-associated lipodystrophy syndrome (HALS) is a side effect of highly active antiretroviral therapy of HIV-infected patients; however, the mechanism of the lipodystrophy and insulin resistance seen in this syndrome remains elusive. Adiponectin, an adipocyte-specific protein, is thought to play an important role in regulating insulin sensitivity. We investigated circulating levels and gene expression of adiponectin in subcutaneous abdominal adipose tissue (AT) from 18 HIV-infected patients with HALS compared with 18 HIV-infected patients without HALS. Implications of cytokines for adiponectin levels were investigated by determining circulating levels of TNF-alpha, IL-6, and IL-8 as well as gene expression of these cytokines in AT. HALS patients exhibited 40% reduced plasma adiponectin levels (P < 0.05) compared with non-HALS subjects. Correspondingly, adiponectin mRNA levels in AT were reduced by >50% (P = 0.06). HALS patients were insulin resistant, and a positive correlation was found between plasma adiponectin and insulin sensitivity (r = 0.55, P < 0.01) and percent limb fat (r = 0.61, P < 0.01). AT mRNA of TNF-alpha, IL-6, and IL-8 was increased in AT of HALS subjects (P < 0.05), and both AT TNF-alpha mRNA and plasma TNF-alpha were negatively correlated to plasma adiponectin (P < 0.05). Finally, TNF-alpha was found in vitro to inhibit human AT adiponectin mRNA by 80% (P < 0.05). In conclusion, HALS patients have reduced levels of plasma adiponectin and adiponectin mRNA in AT. Increased cytokine mRNA in AT is hypothesized to exert an inhibitory effect on adiponectin gene expression and, consequently, to play a role in the reduced plasma adiponectin levels found in HALS patients.     

Physiological role of peroxisomal beta-oxidation in liver of fasted rats

In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal beta-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.     

Effects of physical exercise on liver ATP levels in fasted and phosphate-injected rats

The purpose of the present study was to investigate the effects of exercise (30 min, 23 m/min, 0% grade) on the hepatic levels of ATP in fasted adrenodemedullated rats, with an intraperitoneal injection of sodium phosphate (Na (2) PO (4 ), 0.91 mM) or saline (NaCl). Sodium phosphate was injected to determine if the postulated decrease in liver ATP during exercise may be changed by providing an excess of phosphate. At the end of exercise, a piece of liver was rapidly freeze clamped and used for the enzymatic determination of ATP levels. Liver ATP, in saline-injected rats, was significantly (P < 0.05) decreased by fasting, compared to fed rats (𝒳 +/- SE: 3. 21 +/- 0.2 vs 2.86+/- 0.2 micromol/g). Exercise in fasted rats decreased even more the ATP response in liver (2.58 +/- 0.14 micromol/g). Injection of Na (2) PO (4) did not significantly (P > 0. 05) alter the pattern of ATP response following these 3 conditions (3.35 +/- 0.14 vs 3.0 +/-0.12 vs 2.57 +/- 0.1 micromol/g), ATP levels being significantly (P <0.05) decreased by the fast and the exercise in the fasted state. Fasting and exercise resulted in a significant (P < 0.05) decrease in liver glycogen and plasma glucose concentrations and an increase in free fatty acid levels in both NaCl- and Na (2 )PO (4) -injected groups. In both injection conditions, beta-hydroxybutyrate and peripheral insulin concentrations were respectively, increased and decreased (P < 0.05) by fasting, while norepinephrine and portal glucagon were decreased (P > 0.05) following exercise. The main effect of the injection of Na ( 2) PO (4) was a stimulation (P < 0.05) of peripheral glucagon response following exercise. It is concluded that exercise results in a decrease in liver ATP levels even in fasted rats and that this decrease is not corrected by Na (2 )PO( 4) administration. The decreased liver ATP levels might be involved in the metabolic adaptations to exercise.     

The ghrelin pentapeptide inhibits the secretion of pancreatic juice in rats.

Ghrelin, a 28 amino acids polypeptide was recognized as an endogenous ligand for the growth hormone secretagogue receptor. It turned out that the entire sequence of ghrelin is not necessary for performing the above-mentioned functions. It was suggested that 5 residues (Gly-Ser-Ser(n-octanoyl)-Phe, pentaghrelin) constituted functionally active part of the full-length polypeptide. Ghrelin-28 was found to inhibit pancreatic enzyme output in rats, though the effect of pentaghrelin was not studied so far. The study aimed to determine the involvement of pentaghrelin in pancreatic juice secretion in anaesthetized rats. Male Wistar rats (220 +/- 20 g body weight, b. wt.) were anesthetized, the external jugular vein and common biliary-pancreatic duct were cannulated. Pentaghrelin boluses (i.v., 1.2, 12, and 50 nmol kg(-1) b. wt.) were injected every 30 min with or without CCK-8 infusion, duodenal mucosal CCK(1) receptor blockade with tarazepide, vagotomy and capsaicin pretreatment. Pentaghrelin boluses reduced the volume of pancreatic-biliary juice, protein and trypsin outputs both under basal and CCK-8-stimulated conditions in a dose-dependent manner. However, exogenous pentaghrelin failed to affect the pancreatic secretion in rats subjected to vagotomy, capsaicin deactivation of afferents or pretreatment with Tarazepide. In conclusion, pentaghrelin may control exocrine pancreas secretion by affecting duodenal neurohormonal mechanism(s) involving CCK and vagal nerves in rats.     

Chronic ethanol feeding inhibits plasma levels of insulin-like growth factor-1

The purpose of this study was to determine whether the generalized catabolic effects of chronic ethanol may be associated with a decline in plasma levels of insulin-like growth factor-1 (IGF-1). Male Sprague-Dawley rats were fed a liquid diet containing 5% ethanol or pair-fed a diet made isocaloric with maltose-dextrin. Animals were maintained on this diet for either 12 days or 4.5 months. Another group of animals were fed control diet ad libitum for 2 weeks. After 12 days of feeding, plasma concentrations of IGF-1 in ad libitum fed rats were 771 +/- 41 ng/ml which was greater than concentrations in either pair-fed (595 +/- 23 ng/ml) or ethanol-fed (680 +/- 40 ng/ml) rats (P less than 0.05). After 4.5 months of feeding, plasma levels of IGF-1 in ad libitum and pair-fed rats were similar to the 12 day study (736 +/- 56 and 607 +/- 26 ng/ml, respectively). However, a significant decrease in plasma levels of IGF-1 was observed in ethanol-fed animals over the 4.5 month period (551 +/- 28 ng/ml, P less than 0.05). Results of a similar study in rats fed a high-fat diet for 4.5 months were similar to those found with the low-fat diet. These results indicate that 1) dietary restriction of the type routinely used in this pair-feeding regimen decreases plasma levels of IGF-1, 2) chronic ethanol feeding further decreases plasma IGF-1 levels compared to pair-fed rats, 3) the effects of ethanol on IGF-1 concentrations are not modified by dietary fat, and 4) the effects on IGF-1 are not directly dependent on elevated plasma ethanol concentrations. Our results suggest that IGF-1 secreting cells in the liver may be progressively damaged by chronic ethanol feeding.     

Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

Introduction

Although IL-1β is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1β blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1β signaling in chondrocytes.

Methods

Cartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture. SOCS1 expression in cartilage was assessed with immunohistochemistry. IL-1β-induced SOCS1 expression in chondrocytes was analyzed with quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1β signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1-overexpressing or -knockdown chondrocytes.

Results

SOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P < 0.01). IL-1β stimulated SOCS1 mRNA expression in a dose-dependent pattern (P < 0.01). The IL-1β-induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 overexpression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P values < 0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) activation, and by downregulating transforming growth factor-β-activated kinase 1 (TAK1) expression.

Conclusions

Our results show that SOCS1 is induced by IL1-β in OA chondrocytes and suppresses the IL-1β-induced synthesis of matrix-degrading enzymes by inhibiting IL-1β signaling at multiple levels. It suggests that the IL-1β-inducible SOCS1 acts as a negative regulator of the IL-1β response in OA cartilage.     

Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting as an IL-1 functional antagonist

We have designed a series of recombinant CAT genes to study IL-1 signal transduction in murine fibroblast NIH 3T3 cells. We demonstrate that the HSV thymidine kinase (tk) promoter does not respond to IL-1, but that IL-1 induction of this promoter is observed after insertion of either NF-kB or AP-1 binding sites upstream of the HSV tk cap-site. We have studied the effects of indomethacin, dexamethasone and aurothioglucose (which have been used in the treatment of patients affected by rheumatoid arthritis) in the IL-1 inducible CAT assay. We show that aurothioglucose or dexamethasone is able to inhibit IL-1 induced CAT activity whereas a non-steroidal anti-inflammatory drug (indomethacin) is inactive. Order of addition experiments indicate that aurothiglucose, which has disease-modifying activity in treated patients, ats as an IL-1 functional antagonist in this system.     

Aging influences the level and functions of fasting plasma ghrelin levels: the POWIRS-Study

OBJECTIVE: Ghrelin, known for its orexigenic activity, also have functions such as vasodilation and a growth hormone releasing action. It is uncertain whether these functions change with increasing age. This study aimed to determine whether ghrelin levels differ between young and older women with different levels of obesity; and secondly whether the associations of ghrelin with metabolic syndrome (MS) components, adipocytokines, coagulation factors, and cortisol change with increasing age. METHODS AND RESULTS: Caucasian women (N=107) were divided into young (19-29 years) and older groups (30-56 years). Fasting ghrelin, leptin, adiponectin, glucose, insulin, cortisol, fibrinogen and plasminogen activator inhibitor-1 (PAI-1) levels were determined. Blood pressure (BP), body mass index and waist circumferences were measured. Older lean women showed lower levels of ghrelin (p<0.05) than young lean women, with no differences regarding BP, obesity, lipids, adipokines or insulin resistance (IR). Ghrelin levels of older women remained constant with increasing obesity, but younger women showed significantly reduced ghrelin levels in obese groups. Only younger women showed significant correlations between ghrelin and leptin, adiponectin, fibrinogen and PAI-1 (adjusted for age, obesity and menstrual phase), whereas both age groups showed significant correlations with IR. In younger women factor analysis grouped ghrelin with coagulation factors and all MS components. In older women ghrelin was absent from the MS cluster, but was associated with lower BP, cortisol and IR. CONCLUSIONS: Ghrelin levels were not significantly elevated in lean older women, and did not change with increased obesity in older women--as were observed in younger women. The functions of ghrelin also seem to change with increased age since only in young women ghrelin was associated with obesity, coagulation factors and leptin.     

Colonic production and expression of IL-4, IL-6, and IL-10 in neonatal suckling rats after LPS challenge

It has been demonstrated that the neonatal suckling rat is more susceptible to endotoxin [lipopolysaccharide (LPS)]-induced colonic damage compared with weaned littermates. There is evidence to suggest that differences in the production of certain cytokines, including interleukin (IL)-4, IL-6, and IL-10, are associated with intestinal inflammation in children. We have examined the production, localization, and mRNA detection of these cytokines in suckling and weaned rat colons after bacterial LPS challenge. Suckling (10 day old) and weaned (25 day old) rats were injected with LPS (3 mg/kg ip). Colon samples were taken up to 4 h after treatment, and cytokines were measured by ELISA. LPS-induced cytokine levels were significantly different in suckling rats compared with weaned rats. Cytokine localization to the colonic mucosa was evident in suckling rats up to 4 h after LPS administration but was not consistently seen in weaned rats. The mRNA for cytokines examined were detected by RT-PCR in suckling but not in weaned rat colons after LPS treatment. Induction of neutropenia via anti-neutrophil serum (ANS) administration did not affect cytokine mRNA detection in neonates after LPS treatment. Weaned animals displayed positive detection of all cytokines examined after ANS. Therefore, we have shown that the suckling rat displays a different production and expression of colonic IL-4, IL-6, and IL-10 compared with weaned littermates after LPS challenge. Furthermore, neutrophils may be implicated in colonic cytokine expression after LPS challenge in rats.     

Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting as an IL-1 functional antagonist.

We have designed a series of recombinant CAT genes to study IL-1 signal transduction in murine fibroblast NIH 3T3 cells. We demonstrate that the HSV thymidine kinase (tk) promoter does not respond to IL-1, but that IL-1 induction of this promoter is observed after insertion of either NF-kB or AP-1 binding sites upstream of the HSV tk cap-site. We have studied the effects of indomethacin, dexamethasone and aurothioglucose (which have been used in the treatment of patients affected by rheumatoid arthritis) in the IL-1 inducible CAT assay. We show that aurothioglucose or dexamethasone is able to inhibit IL-1 induced CAT activity whereas a non-steroidal anti-inflammatory drug (indomethacin) is inactive. Order of addition experiments indicate that aurothioglucose, which has disease-modifying activity in treated patients, acts as an IL-1 functional antagonist in this system.     

A comparison of leptin and ghrelin levels in plasma and saliva of young healthy subjects

In the last 10 years, saliva has been increasingly used as a diagnostic fluid and in predictions of disease progression. Leptin and ghrelin are synthesized in several tissues including the salivary glands. The action of ghrelin is antagonistic to that of leptin. This study was undertaken to measure and compare the saliva ghrelin-leptin and plasma ghrelin-leptin levels in healthy young subjects. In 30 healthy subjects, after an overnight fast, saliva and plasma leptin levels were measured using the ELISA method while saliva and plasma immunoreactive ghrelin levels were measured using a commercial radioimmunoassay (RIA). The latter uses 125I-labeled bioactive ghrelin as a tracer and a rabbit polyclonal antibody raised against full-length octanoylated human ghrelin (Phoenix, Europe, Karlsruhe, Germany). The results of this investigation revealed that saliva leptin levels (6.19+/-2.10 microg/l) were lower than plasma levels (7.39+/-3.23 microg/l) while saliva ghrelin levels (188.5+/-84.7 pg/ml) were higher than plasma levels (126.4+/-38.5 pg/ml), when male and female subjects were considered together. Saliva leptin levels (5.93+/-1.94 microg/l) were lower than plasma levels (6.22+/-2.92 pg/ml) while saliva ghrelin levels (190.3+/-80.2 pg/ml) were higher than plasma levels (120.4+/-35.7 pg/ml) in young males. Saliva leptin levels (6.47+/-2.29 microg/l) were lower than plasma levels (8.73+/-3.14 microg/l) while saliva ghrelin levels (183.2+/-90.2 pg/ml) were higher than plasma levels (129.3+/-42.8 pg/ml) in young females, and both saliva and plasma leptin levels were slightly lower in male subjects in comparison with female subjects. Also, Immunohistochemistry study indicated that ghrelin positivity was found in ductus epithelium of salivary gland. We have demonstrated for the first time that saliva ghrelin levels were higher than in plasma while saliva leptin levels were almost the same as in plasma. Measurements of ghrelin and leptin in saliva is non-invasive, simple, and generally much preferred by patients and thus may be an acceptable alternative to plasma sampling.