Altered bioavailability of epoxyeicosatrienoic acids is associated with conduit artery endothelial dysfunction in type 2 diabetic patients.

BACKGROUND: This pathophysiological study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes the vasodilator and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), contributes to conduit artery endothelial dysfunction in type 2 diabetes. METHODS AND RESULTS: Radial artery endothelium-dependent flow-mediated dilatation in response to hand skin heating was reduced in essential hypertensive patients (n = 9) and type 2 diabetic subjects with (n = 19) or without hypertension (n = 10) compared to healthy subjects (n = 36), taking into consideration cardiovascular risk factors, flow stimulus and endothelium-independent dilatation to glyceryl trinitrate. Diabetic patients but not non-diabetic hypertensive subjects displayed elevated whole blood reactive oxygen species levels and loss of NO release during heating, assessed by measuring local plasma nitrite variation. Moreover, plasma levels of EET regioisomers increased during heating in healthy subjects, did not change in hypertensive patients and decreased in diabetic patients. Correlation analysis showed in the overall population that the less NO and EETs bioavailability increases during heating, the more flow-mediated dilatation is reduced. The expression and activity of sEH, measured in isolated peripheral blood mononuclear cells, was elevated in diabetic but not hypertensive patients, leading to increased EETs conversion to DHETs. Finally, hyperglycemic and hyperinsulinemic euglycemic clamps induced a decrease in flow-mediated dilatation in healthy subjects and this was associated with an altered EETs release during heating. CONCLUSIONS: These results demonstrate that an increased EETs degradation by sEH and altered NO bioavailability are associated with conduit artery endothelial dysfunction in type 2 diabetic patients independently from their hypertensive status. The hyperinsulinemic and hyperglycemic state in these patients may contribute to these alterations. Trial registration NCT02311075. Registered December 8, 2014.

Enteral glutamine infusion modulates ubiquitination of heat shock proteins, Grp-75 and Apg-2, in the human duodenal mucosa.

Glutamine, the most abundant amino acid in the human body, plays several important roles in the intestine. Previous studies showed that glutamine may affect protein expression by regulating ubiquitin-proteasome system. We thus aimed to evaluate the effects of glutamine on ubiquitinated proteins in human duodenal mucosa. Five healthy male volunteers were included and received during 5 h, on two occasions and in a random order, either an enteral infusion of maltodextrins alone (0.25 g kg(-1) h(-1), control), mimicking carbohydrate-fed state, or maltodextrins with glutamine (0.117 g kg(-1) h(-1), glutamine). Endoscopic duodenal biopsies were then taken. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using anti-ubiquitin antibody. Differentially ubiquitinated proteins were then identified by liquid chromatography-electrospray ionization MS/MS. Five proteins were differentially ubiquitinated between control and glutamine conditions. Among these proteins, we identified two chaperone proteins, Grp75 and hsp74. Grp75 was less ubiquitinated after glutamine infusion compared with control. In contrast, hsp74, also called Apg-2, was more ubiquitinated after glutamine. In conclusion, we provide evidence that glutamine may regulate ubiquitination processes of specific proteins, i.e., Grp75 and Apg-2. Grp75 has protective and anti-inflammatory properties, while Apg-2 indirectly regulates stress-induced cell survival and proliferation through interaction with ZO-1. Further studies should confirm these results in stress conditions.

The effect of camelina oil on vascular function in essential hypertensive patients with metabolic syndrome: a randomized, placebo-controlled, double-blind study.

BACKGROUND: The effects of a dietary supplementation with the vegetable ω-3 α-linolenic acid (ALA) on cardiovascular homeostasis are unclear. In this context, it would be interesting to assess the effects of camelina oil. OBJECTIVE: This study aimed to assess the cardiovascular and metabolic effects of camelina oil in hypertensive patients with metabolic syndrome. METHODS: In a double-blind, placebo-controlled randomized study, treated essential hypertensive patients with metabolic syndrome received, during 6 mo, either cyclodextrin-complexed camelina oil containing ≈ 1.5 g ALA/d (n = 40) or an isocaloric placebo (n = 41), consisting of the same quantity of cyclodextrins and wheat starch. Anthropometric data, plasma lipids, glycemia, insulinemia, creatininemia, TBARs, high-sensitivity C-reactive protein, and n-3, n-6, and n-9 fatty acids in erythrocyte membranes were measured. Peripheral and central blood pressures, arterial stiffness, carotid intima-media thickness, and brachial artery endothelium-dependent flow-mediated dilatation (FMD) and endothelium-independent dilatation were assessed. RESULTS: Compared with placebo, camelina oil increased ALA (mean ± SD: 0 ± 0.04 compared with 0.08 ± 0.06%, P <0.001), its elongation product EPA (0 ± 0.5 compared with 0.16 ± 0.65%, P <0.05), and the n-9 gondoic acid (GA; 0 ± 0.04 compared with 0.08 ± 0.04%, P <0.001). No between-group difference was observed for cardiovascular parameters. However, changes in FMD were associated with the magnitude of changes in EPA (r = 0.26, P = 0.03). Compared with placebo, camelina oil increased fasting glycemia (-0.2 ± 0.6 compared with 0.3 ± 0.5 mmol/L, P <0.001) and HOMA-IR index (-0.8 ± 2.5 compared with 0.5 ± 0.9, P <0.01), without affecting plasma lipids, or inflammatory and oxidative stress markers. Changes in HOMA-IR index were correlated with the magnitude of changes in GA (r = 0.32, P <0.01). Nutritional intake remained similar between groups. CONCLUSION: ALA supplementation with camelina oil did not improve vascular function but adversely affected glucose metabolism in hypertensive patients with metabolic syndrome. Whether this adverse effect on insulin sensitivity is related to GA enrichment, remains to be elucidated.

The neuropeptide substance P regulates aldosterone secretion in human adrenals.

Aldosterone, produced by the adrenals and under the control of plasma angiotensin and potassium levels, regulates hydromineral homeostasis and blood pressure. Here we report that the neuropeptide substance P (SP) released by intraadrenal nerve fibres, stimulates aldosterone secretion via binding to neurokinin type 1 receptors (NK1R) expressed by aldosterone-producing adrenocortical cells. The action of SP is mediated by the extracellular signal-regulated kinase pathway and involves upregulation of steroidogenic enzymes. We also conducted a prospective proof-of-concept, double blind, placebo-controlled clinical trial aimed to investigate the impact of the NK1R antagonist aprepitant on aldosterone secretion in healthy male volunteers (EudraCT: 2008-003367-40, ClinicalTrial.gov: NCT00977223). Participants received during two 7-day treatment periods aprepitant (125 mg on the 1st day and 80 mg during the following days) or placebo in a random order at a 2-week interval. The primary endpoint was plasma aldosterone levels during posture test. Secondary endpoints included basal aldosterone alterations, plasma aldosterone variation during metoclopramide and hypoglycaemia tests, and basal and stimulated alterations of renin, cortisol and ACTH during the three different stimulatory tests. The safety of the treatment was assessed on the basis of serum transaminase measurements on days 4 and 7. All pre-specified endpoints were achieved. Aprepitant decreases aldosterone production by around 30% but does not influence the aldosterone response to upright posture. These results indicate that the autonomic nervous system exerts a direct stimulatory tone on mineralocorticoid synthesis through SP, and thus plays a role in the maintenance of hydromineral homeostasis. This regulatory mechanism may be involved in aldosterone excess syndromes.

Characterization of the EM66 Biomarker in the Pituitary and Plasma of Healthy Subjects With Different Gonadotroph Status and Patients With Gonadotroph Tumor.

Granins and their derived-peptides are useful markers of secretion from normal and tumoral neuroendocrine cells. The need to identify new diagnostic markers for neuroendocrine tumors, including pituitary tumors prompted us to determine plasma levels of the secretogranin II-derived peptide EM66 in healthy volunteers with different gonadotroph status and to evaluate its usefulness as a circulating marker for the diagnosis of gonadotroph tumor. Using a radioimmunoassay, we determined plasma EM66 concentrations in healthy men and women volunteers in different physiological conditions in relation with the gonadotroph function. Our results revealed that in men, in women with or without contraception, in pregnant or post-menopausal women, plasma EM66 concentrations are not significantly different, and did not show any correlation with gonadotropin levels. In addition, stimulation or inhibition tests of the gonadotroph axis had no effect on EM66 levels, whatever the group of healthy volunteers investigated while gonadotropin levels showed the expected variations. Immunohistochemical experiments and HPLC analysis showed the occurrence of EM66 in pituitary gonadotroph, lactotroph and corticotroph tumors but not in somatotroph tumor. In patients with gonadotroph or lactotroph tumor, plasma EM66 levels were 1.48 (0.82-4.38) ng/ml and 2.49 (1.19-3.54) ng/ml, respectively. While median value of EM66 was significantly lower in patients with gonadotroph tumor compared to healthy volunteers [2.59 (0.62-4.95) ng/ml], plasma EM66 concentrations were in the same range as normal values and did not show any correlation with gonadotropin levels. These results show that plasma EM66 levels are independent of the activity of the gonadotroph axis in healthy volunteers and, while EM66 levels are reduced in gonadotroph tumors, plasma EM66 does not provide a helpful marker for the diagnosis of these tumors.

Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

BACKGROUND: Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. OBJECTIVE: We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. DESIGN: Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. RESULTS: Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and ß-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). CONCLUSIONS: Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling.

Characteristics of follicular cell-derived thyroid carcinomas occurring after external radiation exposure: results of a case control study nested in a cohort.

Radiation exposure is the only well-established risk factor for follicular cell-derived thyroid carcinoma. To compare the clinical characteristics and outcome of thyroid carcinoma in patients with and without a history of radiation exposure, we performed a case control study nested in a cohort of 2,196 patients treated for a papillary or a follicular thyroid cancer at the Institut Gustave Roussy. The study was performed on 91 cases (71% females) and their 273 controls matched for gender, age at thyroid cancer diagnosis (+/-3 years), and period of initial thyroid cancer treatment (+/-6 years). More than 85% of the cases have been first exposed to external radiation before the age of 30 years. Thyroid cancers were more frequently multifocal in cases than in controls (odds ratio [OR] 2.5, 95% confidence interval [CI] 1.3-4.8), and local residual tumor was more frequently observed in cases, but the other clinical features did not differ overall. Female cases with a history of radiation exposure more frequently had a tumor of follicular histology than female controls (OR = 2.7, 95% CI: 1.1-6.5), and conversely the frequency of follicular histology was similar in male cases and in male controls (OR = 0.3, 95% CI: 0.1-1.4). The risks of recurrence and of thyroid cancer related death were similar in cases and in controls.

Relationship between tumor burden and serum thyroglobulin level in patients with papillary and follicular thyroid carcinoma.

Serum thyroglobulin (Tg) is a reliable marker for detecting recurrent and persistent disease during the follow-up of patients with papillary and follicular thyroid carcinoma. The goal of this study was to assess the relationship between the serum Tg level measured after thyroid hormone withdrawal and the tumor mass in thyroid cancer patients who underwent surgery with the use of an intraoperative probe for lymph node metastases with (131)I uptake. Patients were classified into one of three groups according to the Tg level: undetectable (n = 18); 1-10 ng/mL (n = 21); and greater than 10 ng/mL (n = 33). The main clinical characteristics and the extent of the disease at the time of initial treatment were similar in these three groups. Lymph node metastases were found in 13 of the 18 patients with undetectable Tg level. Eight patients had persistent foci of uptake after surgery that were located behind the sterno-clavicular joint in six patients. The number of metastatic lymph nodes and their total surface (in mm(2)) or their total volume (in mm(3)) were significantly linked with serum Tg/thyrotropin [TSH] level (p = 0.002 and p < 0.0001, respectively). For a given metastatic surface or volume, the serum Tg/TSH value was no longer linked with the number of metastatic lymph nodes (p = 0.32), suggesting that the total surface or total volume is the characteristic that best summarizes the influence of the disease on the serum Tg/TSH level. In conclusion, patients with higher serum Tg levels tend to have more extensive disease and should undergo more aggressive treatment modalities. Nevertheless, undetectable serum Tg should not be considered as a reliable criteria to exclude a minimal tumor burden in patients who have already been treated with (131)I.

Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin-independent pathway.

BACKGROUND: Glutamine modulates duodenal protein metabolism in fasted healthy humans, but its effects in a fed state remain unknown. OBJECTIVE: We aimed to assess the effects of either glutamine or an isonitrogenous protein mixture on duodenal protein metabolism in humans in the fed state. DESIGN: Twenty-four healthy volunteers were randomly included in 2 groups. Each volunteer was studied on 2 occasions in a random order and received, during 5 h, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹; both groups) that mimicked a carbohydrate fed state or maltodextrins with glutamine (group 1) or an isonitrogenous (22.4 mg N · kg⁻¹ · h⁻¹) protein powder (group 2). Simultaneously, a continuous intravenous infusion of ¹³C-leucine and ²H5-phenylalanine (both 9 µmol · kg⁻¹ · h⁻¹) was performed. Endoscopic duodenal biopsies were taken. Leucine and phenylalanine enrichments were assessed by using gas chromatography-mass spectrometry in duodenal proteins and the intracellular free amino acids pool to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates and macroarrays, respectively. RESULTS: The FSR and proteasome activity were not different after the glutamine supply compared with after maltodextrins alone. In contrast, the FSR increased (1.7-fold increase; P < 0.05) after protein-powder delivery without modification of total proteasome activity. The protein powder increased insulinemia, PI3 kinase, and erk phosphorylation but did not affect the mammalian target of rapamycin (mTOR) pathway and mitogen-activated protein kinase signal-integrating kinase 1 phosphorylation. A trend for an increase of eukaryotic translation initiation factor 4E phosphorylation was observed (P = 0.07). CONCLUSION: In the carbohydrate fed state, enteral proteins but not glutamine increased duodenal protein synthesis through an mTOR independent pathway in humans.

An enteral leucine supply modulates human duodenal mucosal proteome and decreases the expression of enzymes involved in fatty acid beta-oxidation.

Leucine is well known to regulate protein metabolism in muscle. We recently reported that enteral leucine infusion decreased proteasome activity in human duodenal mucosa and enhanced intestinal cell proliferation, but its effects on gut proteome remain unknown. Therefore, we aimed to assess the effects of an enteral leucine infusion on the whole proteome of duodenal mucosa. In this work, 5 healthy volunteers received for 5h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g kg(-1) h(-1)) or maltodextrins supplemented with leucine (0.035 g kg(-1) h(-1)). At the end of infusion, endoscopic duodenal biopsy samples were collected and analyzed by 2D-PAGE. Eleven protein spots were differentially and significantly (P<0.05) expressed in response to the leucine-supplemented maltodextrins compared with maltodextrins alone. Forty percent of identified proteins by mass spectrometry were located in mitochondria. Four proteins were involved in lipid metabolism: HADHA, ACADVL and CPT2 expressions were reduced, whereas FABP1 expression was increased. In addition, the expression of DHA kinase involved in glycerol metabolism was also downregulated. Finally, leucine supplementation altered the duodenal mucosal proteome by regulating the expression of several enzymes mainly involved in lipid metabolism. These results suggest that leucine supplementation may slowdown fatty acid beta-oxidation in human duodenal mucosa.

Influence of leucine on protein metabolism, phosphokinase expression, and cell proliferation in human duodenum1,3.

BACKGROUND: Although leucine increases protein anabolism through the mammalian target of rapamycin (mTOR) pathway in human muscles, its effects on intestinal mucosal proteins remain unknown. OBJECTIVE: We aimed to assess the effects of leucine on duodenal protein metabolism in healthy humans and to elucidate the signaling pathways involved. DESIGN: Eleven healthy volunteers received for 5 h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g . kg(-1) . h(-1)) or maltodextrins and leucine (0.035 g . kg(-1) . h(-1)) simultaneously with a continuous intravenous infusion of [(2)H(5)]phenylalanine (9 µmol . kg(-1) .h(-1)). Endoscopic duodenal biopsy samples were collected and frozen until analyzed. Phenylalanine enrichment was assessed by gas chromatography-mass spectrometry in duodenal protein and in free intracellular amino acid pools used as precursor to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates or macroarrays, respectively. RESULTS: Leucine supplementation slightly reduced FSR (mean ± SEM: 81.3 ± 6.3%/d) compared with maltodextrins alone (91.7 ± 8.5%/d; P = 0.0537). In addition, total proteasome activity decreased significantly with leucine (236 ± 21 compared with 400 ± 58 relative fluorescence units/µg protein; P < 0.05), with no modification of chymotrypsin-like, trypsin-like, caspase-like, or peptidase activities. Leucine did not affect the mTOR pathway but did increase the phosphorylation states of PI3K, Akt, AMPK, p38 MAPK, JNK, GSK-3α/ß, STAT3, and STAT5 and increased cyclin D1 mRNA concentrations, which suggested that leucine may enhance cell proliferation. CONCLUSION: Enteral leucine supplementation decreased proteasome activity in duodenal mucosa and enhanced cell proliferation through the PI3K/Akt/GSK-3α/ß-catenin pathway. This trial was registered at clinicaltrials.gov as NCT01254110.

Effects of an enteral glucose supply on protein synthesis, proteolytic pathways, and proteome in human duodenal mucosa.

BACKGROUND: Previous studies have shown that the glucose supply reduces postoperative insulin resistance and improves patient outcomes. However, the effects of luminal glucose on intestinal mucosal proteins remain unknown. OBJECTIVE: We aimed to assess the effects of an enteral glucose supply on protein synthesis, proteolytic pathways, and proteome in human duodenal mucosa. DESIGN: Twenty healthy volunteers received a 5-h enteral infusion of either saline or glucose (0.12 g · kg(-1) · h(-1)). Simultaneously, a continuous intravenous infusion of l-[1-(13)C]leucine (12 µmol · kg(-1) · h(-1)) was maintained until endoscopy. The duodenal mucosal protein fractional synthesis rate (FSR) was calculated from leucine enrichments assessed in protein and free amino acid pools by gas chromatography-mass spectrometry. Cathepsin D, calpains, and chymotrypsin-like proteasome mucosal activities were evaluated by using specific fluorogenic substrates. A 2-dimensional PAGE-based comparative proteomics analysis was also performed on additional duodenal mucosal biopsy samples to identify differentially expressed proteins. RESULTS: Duodenal mucosal protein FSR and protease activities were not affected by glucose infusion relative to saline. Nevertheless, the comparative proteomics analysis indicated that 10 protein spots were significantly differentially expressed (ie, at least ±1.5-fold modulated; Student's t test, P < 0.05) in response to the glucose infusion relative to saline. Of the 8 proteins identified by mass spectrometry, α-enolase, cytoplasmic aconitate hydratase, and glutathione S-transferase ω-1 were upregulated, whereas epoxide hydrolase 2 was downregulated. CONCLUSION: Enteral glucose supply affected neither duodenal mucosal protein FSR nor activities of mucosal proteases but altered the duodenal mucosal proteome by modulating the expression of several enzymes involved mainly in carbohydrate and xenobiotic metabolism. This trial is registered at clinicaltrials.gov as NCT00213551.