Peter C. Konturek

Leptin and ghrelin levels in patients with obstructive sleep apnoea: effect of CPAP treatment

Serum leptin and ghrelin levels were investigated in patients with obstructive sleep apnoea (OSA) syndrome before and during continuous positive airways pressure (CPAP) treatment and compared with body mass index (BMI)-matched controls without OSA. Male patients (n=30) with OSA (apnoea/hypopnoea index=58±16, BMI=32.6±5.3 kg·m−2) underwent CPAP treatment. Fasting leptin and ghrelin were measured atbaseline and 2 days, and in the case of leptin 2 months after initiation of treatment. Baseline plasma ghrelin levels were significantly higher in OSA patients than in controls. After 2 days of CPAP treatment, plasma ghrelin decreased in almost all OSA patients (n=9) to levels that were only slightly higher than those of controls (n=9). Leptin levels did not change significantly from baseline after 2 days of CPAP treatment, but were higher than in the control group. After 8 weeks, leptin levels decreased significantly, although the BMI of the patients showed no change. The decrease in leptin levels was more pronounced in patients with a BMI <30 kg·m−2. These data indicate that the elevated leptin and ghrelin levels are not determined by obesity alone, since they rapidly decreased during continuous positive airways pressure therapy.

The Effect of Continuous Positive Airway Pressure Treatment on Insulin Sensitivity in Patients with Obstructive Sleep Apnoea Syndrome and Type 2 Diabetes

Background: The obstructive sleep apnoea syndrome (OSA) is a frequent condition, as well as type 2 diabetes mellitus. Both diseases are characterized by insulin resistance. Objectives: The aim of this study was to establish whether OSA is an independent risk factor for increased insulin resistance in diabetics. For this purpose, we tested the hypothesis that the insulin sensitivity in patients with type 2 diabetes and OSA can be improved by 2 days or 3 months of continuous positive airway pressure (CPAP) treatment. Methods: In 9 obese patients with type 2 diabetes and OSA [apnoea/hypopnoea index 43.1 ± 21.3; body mass index (BMI) 37.3 ± 5.6 kg/m2] and good glycaemic control on oral antidiabetics or on diet alone (HbA1c 6.4 ± 0.7%), the insulin sensitivity index (ISI) was established by euglycaemic hyperinsulinaemic clamp tests at baseline, after 2 days and after 3 months of effective CPAP treatment. Results: ISI was unchanged after 2 days of CPAP treatment, but was significantly improved after 3 months (4.38 ± 2.94 vs. 2.74 ± 2.25 at baseline; p = 0.021), without any significant changes in BMI. Glycaemic control was unaffected after 3 months (HbA1c 6.3 ± 0.6%; not significant). Fasting leptin levels showed no significant changes. Conclusions: These results indicate that OSA itself is an independent risk factor for insulin resistance. This effect may be explained by the elevated sympathetic activity in OSA.

Activation of NFκB Represents the Central Event in the Neoplastic Progression Associated with Barrett's Esophagus: A Possible Link to the Inflammation and Overexpression of COX-2, PPARγ and Growth Factors

The molecular mechanisms responsible for the progression of malignant transformation in Barretts esophagus (BE) are still poorly understood. This study was undertaken (1) to investigate the gene and protein expression of cyclooxygenase-2 (COX-2), peroxisome proliferator-activated receptor-gamma (PPARγ), interleukin-8 (IL-8), hepatocyte growth factor (HGF), gastrin, and its receptor (CCK-2) in the Barretts epithelium; (2) to analyze the activity of NFκB in Barretts esophagus with low-grade dysplasia; and (3) to assess the effects of PPARγ ligand (ciglitazone) and gastrin on cell proliferation in the cell line derived from esophageal adenocarcinoma (OE-33). COX-2, PPARγ, IL-8, HGF, gastrin, and CCK-2 expression levels relative to the control gene encoding GAPDH were analyzed by RT-PCR and Western blot in specimens of BE with low-grade dysplasia (n = 20) and compared with that in the normal squamous esophageal mucosa from the middle portion of the esophagus (n = 20). In vitro experiments included the incubation of cell line OE-33 with ciglitazone (1–15 μ M) and gastrin (100 nM). NFκB activity in biopsies specimens was measured by highly sensitive ELISA. COX-2, PPARγ, IL-8, HGF, gastrin, and CCK-2 expressions were significantly increased in BE compared with normal squamous esophageal mucosa. NFκB activity was significantly upregulated in BE. Ciglitazone inhibited cell proliferation of OE-33 cells as assessed by BrdU and this effect was attenuated partly by gastrin. (1) COX-2, PPARγ, HGF, gastrin, and its receptor are significantly upregulated in BE, suggesting a possible role for these factors in Barretts carcinogenesis; (2) the increased NFκB activity is probably linked to increased IL-8 and COX-2 expression; and (3) PPARγ ligands might be useful as a new therapeutic option in the prevention and treatment of Barretts carcinoma.

Role of leptin in ulcer healing

Leptin was shown to exhibit similar to cholecystokinin (CCK) cytoprotective activity against acute gastric lesions, but its role in ulcer healing has not been examined. The aims of this study were: (1) to compare the effects of exogenous leptin to those of CCK on the course of healing of chronic gastric ulcers; (2) to study the gene and protein expression of leptin at the ulcer margin during ulcer healing; and (3) to assess the effects of leptin administration on the mucosal gene expression of main growth factor such as transforming growth factor alpha (TGFalpha). Gastric ulcers were produced in rats by the acetic acid method. Rats with ulcers were divided in following treatment groups: (1) vehicle; (2) leptin (10 microg/kg i.p.); (3) CCK (10 microg/kg s.c.); and (4) leptin or CCK with or without tyrphostin A46 (200 microg/kg i.p.), an inhibitor of epidermal growth factor (EGF)-receptor tyrosine kinase or NG-nitro-L-arginine (20 mg/kg i.g.), a blocker of nitric oxide synthase. Animals were euthanized 9 days after ulcer induction. The area of gastric ulcers and the gastric blood flow at the ulcer area were determined. In addition, mucosal biopsy samples were taken from the ulcer area for histological evaluation as well as for the determination of mRNA and protein expression for leptin and constitutive nitric oxide synthase (cNOS) and inducibile nitric oxide synthase (iNOS) by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. In addition, the gene expression for TGFalpha was analyzed by RT-PCR. Both leptin and CCK reduced significantly the ulcer area as compared to vehicle-treated group by approximately 50%. The treatment with tyrphostin or N(G)-nitro-L-arginine reversed in part the acceleration of ulcer healing by leptin and CCK. The expression of leptin mRNA and protein was significantly increased at the ulcer edge. The leptin-induced acceleration of ulcer healing was associated with increased expression of transcripts for TGFalpha as well as increased mRNA and protein expression for cNOS and iNOS at the ulcer margin. We conclude that leptin accelerates ulcer healing by mechanisms involving the up-regulation of TGFalpha and increased production of nitric oxide due to up-regulation of cNOS and iNOS in the ulcer area.

Prostaglandin/Cyclooxygenase Pathway in Ghrelin-Induced Gastroprotection against Ischemia-Reperfusion Injury

Ghrelin is involved in the control of food intake, but its role in gastroprotection against the formation of gastric mucosal injury has been little elucidated. We studied the effects of peripheral (i.p.) and central (i.c.v.) administration of ghrelin on gastric secretion and gastric mucosal lesions induced by 3 h of ischemia/reperfusion (I/R) with or without inhibition of ghrelin growth hormone secretagogue type 1a receptor (GHS-R1a) by using ghrelin antagonist, d-Lys3-GHRP-6; blockade of cyclooxygenase (COX)-1 (indomethacin, SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole]) and COX-2 (rofecoxib); and bilateral vagotomy or capsaicin denervation. I/R produced typical gastric erosions, a significant fall in the gastric blood flow (GBF), an increase in gastric myeloperoxidase (MPO) activity and malonyldialdehyde (MDA) content, and the up-regulation of mucosal ghrelin mRNA. Ghrelin dose-dependently increased gastric acid secretion and significantly reduced I/R-induced gastric erosions, while producing a significant rise in the GBF and mucosal PGE2 generation and a significant fall in MPO activity and MDA content. The protective and hyperemic activities of ghrelin were significantly attenuated in rats pretreated with d-Lys3-GHRP-6 and capsaicin denervation and completely abolished by vagotomy. Indomethacin, SC560, and rofecoxib, selective COX-1 and COX-2 inhibitors, attenuated ghrelin-induced protection that was restored by supplying the methyl analog of prostaglandin (PG) E2. The expression of mRNA for COX-1 was unaffected by ghrelin, but COX-2 mRNA and COX-2 protein were detectable in I/R injured mucosa and further up-regulated by exogenous ghrelin. We conclude that ghrelin exhibits gastroprotective and hyperemic activities against I/R-induced erosions, the effects that are mediated by hormone activation of GHS-R1a receptors, COX-PG system, and vagal-sensory nerves.

Expression of survivin and caspase-3 in gastric cancer.

Gastric cancer is one of the most common malignant tumors of the gastrointestinal tract. However, the molecular pathways involved in the regulation of gastric carcinogenesis are not completely elucidated. In the last decade, basic cancer research has been focused on the deregulation of apoptosis as a central event in the process of carcinogenesis. Caspase-3 and survivin are regulators of apoptosis and have been implicated in the development of gastric cancer. The aim of the present study was to compare the expression of mRNA and protein for survivin and caspase-3 in the gastric cancer and in the cancer margin with that in normal human gastric mucosa. Fifteen patients with advanced gastric cancer (all H. pylori-positive) and 15 matched control subjects with normal gastric mucosa were included in this study. The biospy specimens for histology and for molecular analyses were taken from gastric tumor, tumor surrounding gastric mucosa and in normal patients from the mucosa of antrum and corpus. Survivin mRNA expression was very weak, but detectable, in the normal gastric mucosa. However, at the protein level, no expression for survivin was detected in the normal gastric mucosa. In the biopsy specimens from tumor and surrounding gastric mucosa, a significant increase in survivin mRNA and protein expression was observed. The expression of survivin was higher in the tumor than in the tumor margin. The mRNA and protein expression of caspase-3 was detected in the gastric mucosa of normal subjects. In gastric cancer only the expression of procaspase-3 was observed, while the expression of active caspase-3 was completely undetectable. In the gastric mucosa surrounding gastric cancer, no gene and protein expression for caspase-3 was detected. We conclude that the changes in the level of caspase-3 and survivin play an important role in the transformation from normal gastric mucosa to gastric career.

Therapeutic Potential of 1-Methylnicotinamide against Acute Gastric Lesions Induced by Stress : Role of Endogenous Prostacyclin and Sensory Nerves

1-Methylnicotinamide (MNA) is one of the major derivatives of nicotinamide, which was recently shown to exhibit antithrombotic and antiinflammatory actions. However, it is not yet known whether MNA affects gastric mucosal defense. The effects of exogenous MNA were studied on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and water restraint stress (WRS) or in rats administered 75% ethanol. MNA [6.25–100 mg/kg intragastrically (i.g.)] led to a dose-dependent rise in the plasma MNA level, inhibited gastric acid secretion, and attenuated these gastric lesions induced by WRS or ethanol. The gastroprotective effect of MNA was accompanied by an increase in the gastric mucosal blood flow and plasma calcitonin gene-related peptide (CGRP) levels, the preservation of prostacyclin (PGI2) generation (measured as 6-keto-PGF1α), and an overexpression of mRNAs for cyclooxygenase (COX)-2 and CGRP in the gastric mucosa. R-3-(4-Fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO 324479), which is the selective antagonist of IP/PGI2 receptors, reversed the effects of MNA on gastric lesions and GBF. MNA-induced gastroprotection was attenuated by suppression of COX-1 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; SC-560] and COX-2 [4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one; rofecoxib] activity, capsaicin denervation, and by the pretreatment with CGRP8-37 or capsazepine. Addition of exogenous PGI2 or CGRP restored the MNA-induced gastroprotection in rats treated with COX-1 and COX-2 inhibitors or in those with capsaicin denervation. WRS enhanced MDA content while decreasing superoxide dismutase (SOD) activity in the gastric mucosa, but pretreatment with MNA reversed these changes. MNA exerts potent gastroprotection against WRS damage via mechanisms involving cooperative action of PGI2 and CGRP in preservation of microvascular flow, antioxidizing enzyme SOD activity, and reduction in lipid peroxidation.

Helicobacter pylori infection and gastrin and cyclooxygenase expression in gastric and colorectal malignancies

Helicobacter pylori, infecting more than 50% of the world population, results in gastritis, usually located in the antral portion of the stomach, accompanied by hypergastrinemia, the key factor in gastric and colorectal carcinogenesis. Excessive mucosal cell proliferation for many years may eventually result in gastric atrophy, cell mutation and transformation of gastric mucosal cells into gastrin-producing cells, which also express gastrin receptors serving to stimulate cell proliferation and tumor growth. These processes may be completed by the expression of cyclooxygenase-2 (COX-2) as an inflammation enzyme to release excessive amounts of PGE(2), leading to further proliferation, reduction in apoptosis, angiogenesis and tumor growth. H. pylori eradication results in complete regression of MALT lymphoma and subsequent normalisation of excessive gastrin release and COX-2 expression. Reduction of gastrin by active immunisation (gastrimmune), blocking of gastrin receptors with specific blockers and suppression of COX-2 might be helpful in inhibiting tumor growth and invasion.

Central Leptin and Cholecystokinin in Gastroprotection against Ethanol-Induced Damage

Background: Leptin, a product of the ob gene controlling food intake, has recently been detected in the stomach and shown to be released by cholecystokinin (CCK) and to induce gastroprotection against various noxious agents, but it is not known whether centrally applied leptin influences gastric secretion and mucosal integrity. Aims: In this study we compared the effects of leptin and CCK-8 applied intracerebroventricularly (i.c.v.) on gastric secretion and gastric mucosal lesions induced by topical application of 75% ethanol. Methods: Several major series of Wistar rats were used in this study. The effects of leptin or CCK applied i.c.v. on gastric secretion were examined using conscious rats with gastric fistulas. For the studies on gastroprotection the following series of rats were used to determine the effects of: (A) leptin and CCK applied centrally on this protection and the blockade of CCKA with loxiglumide (30 mg/kg i.p.) and CCKB receptors with RPR 102681 (30 mg/kg i.p.); (B) cutting of vagal nerves; (C) inactivation of sensory nerves by capsaicin (125 mg/kg s.c.); (D) inhibition of calcitonin gene-related peptide (CGRP) receptors with CGRP8–37 (100 μg/kg i.p.), and (E) suppression of nitric oxide synthase (NOS) with NG-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.v.) on ethanol-induced gastric lesions in rats with or without the i.c.v. pretreatment with leptin or CCK-8. Rats were anesthetized 1 h after ethanol administration to measure the gastric blood flow (GBF) and then to determine the area of gastric lesions by planimetry. Blood was withdrawn for the measurement of plasma leptin and gastrin levels by radioimmunoassay and gastric biopsy samples were collected for the determination of cNOS and iNOS mRNA by RT-PCR. Results: Leptin and CCK-8 (0.01–5 μg/kg i.c.v.) dose dependently attenuated gastric lesions induced by 75% ethanol; the doses reducing these lesions by 50% (ED50) were 0.8 and 1.2 μg/kg, respectively. The protective effects of leptin and CCK-8 applied i.c.v. were accompanied by a significant rise in plasma leptin level and an increase in GBF. Blockade of CCKA receptors with loxiglumide abolished the protective and hyperemic effects of CCK but not those of leptin, while RPR 10268, a specific antagonist of CCKB receptors, counteracted leptin-induced protection and the rise in the GBF but failed to influence those afforded by CCK-8. For comparison, pretreatment with peripheral CCK-8 or leptin (10 μg/kg i.p.) causing a similar rise in the plasma leptin level also significantly reduced gastric lesions induced by 75% ethanol. The protective and hyperemic effects of centrally administered leptin were abolished by vagotomy, producing a fall in plasma leptin levels, and significantly attenuated by sensory denervation with capsaicin, by pretreatment with the CGRP antagonist, CGRP8–37, or with L-NAME. A strong signal for iNOS mRNA was recorded in the gastric mucosa of leptin- and CCK-8-treated animals, whereas cNOS mRNA was unaffected. Conclusions: (1) Central leptin exerts a potent gastroprotective action at a dose that has no influence on gastric secretion; (2) this protection depends upon CCKB receptors, vagal activity and sensory nerves, and involves hyperemia probably mediated by NO, and (3) leptin mimics the gastroprotective effect of CCK and may be implicated in the protective and hyperemic actions of this peptide on the rat stomach.

Pioglitazone, a specific ligand of peroxisome proliferator-activated receptor-gamma, accelerates gastric ulcer healing in rat

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation and carcinogenesis. We determined the effect of the specific PPAR-gamma ligand, pioglitazone (5-40 mg/kg intragastrically), on the healing of acetic-acid gastric ulcers in rats. At day 8 after ulcer induction, the ulcer area, the gastric blood flow and mucosal expression of proinflammatory cytokines such as interleukin-1beta, tumour necrosis factor alpha (TNF-alpha) and cyclooxygenase-1, cyclooxygenase-2, constitutive nitric oxide synthase (cNOS), inducible nitric oxide synthase (iNOS) and heat shock protein 70 (HSP70) was determined. Pioglitazone reduced the area of gastric ulcers and raised significantly the gastric blood flow at the ulcer margin and downregulated the mRNA for interleukin-1beta, TNF-alpha, cyclooxygenase-2 and iNOS while cyclooxygenase-1 mRNA was not affected. The expression of PPAR-gamma mRNA was increased in the ulcerated gastric mucosa. We conclude that pioglitazone accelerates the healing of preexisting gastric ulcers due to the hyperemia at ulcer margin and the anti-inflammatory action including suppression of interleukin-1beta, TNF-alpha, cyclooxygenase-2 and iNOS and by an overexpression of HSP70.