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The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that regulates essential physiological functions. In particular, activation of GHSR in response to its endogenous agonist ghrelin promotes food intake and blood glucose increase. Therefore, compounds aimed at blocking GHSR signaling constitute potential options against obesity-related metabolic disorders. We have previously developed potent ligands of GHSR based on a triazole scaffold. Here, we report a new 3,4,5-trisubstituted 1,2,4-triazole compound, named JMV 6616, that potently blocks GHSR activity in vitro and in vivo. Specifically, in HEK293T cells JMV 6616 behaves as an inverse agonist since it binds to GHSR and inhibits its ghrelin-independent signaling. Accordingly, using purified labeled GHSR assembled into lipid nanodiscs we found that JMV 6616 decreases GHSR-catalyzed G protein activation and stabilizes an inactive receptor conformation. Importantly, JMV 6616 also acts on native GHSR since it blocks the insulinostatic effect of ghrelin in pancreatic islets. In mice, JMV 6616 inhibits blood glucose-raising effects of ghrelin treatment and the orexigenic actions of acute ghrelin administration. Together, our data suggest that this triazole-derived modulator of GHSR holds promise to mitigate several pathological features associated with eating and metabolic disorders.

Background The hormone ghrelin is the endogenous agonist of the G-protein coupled receptor (GPCR) termed growth-hormone secretagogue receptor (GHSR). Ghrelin inhibits glucose-stimulated insulin secretion by activating pancreatic GHSR. Recently, Liver-Expressed Antimicrobial Peptide 2 (LEAP2) was recognized as an endogenous GHSR ligand that blocks ghrelin-induced actions. Nonetheless, the effect of LEAP2 on glucose-stimulated insulin secretion from pancreatic islets is unknown. Objectives We aimed at exploring the activity of LEAP2 on glucose-stimulated insulin secretion. Methods Islets of Langerhans isolated from rat pancreas were exposed to glucose in the presence or in the absence of LEAP2 and ghrelin and then insulin secretion was assayed. Results LEAP2 did not modulate glucose-stimulated insulin secretion. However, LEAP2 blocked the insulinostatic action of ghrelin. Conclusion Our data show that LEAP2 behaves as an antagonist of pancreatic GHSR.

International audience

International audience; GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4 and 5, 2 this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

Polyphenols exert pharmacological actions through protein-mediated mechanisms and by modulating intracellular signalling pathways. We recently showed that a gut-microbial metabolite of ellagic acid named urolithin C is a glucose-dependent activator of insulin secretion acting by facilitating L-type Ca2+ channel opening and Ca2+ influx into pancreatic β-cells. However, it is still unknown whether urolithin C regulates key intracellular signalling proteins in β-cells. Here, we report that urolithin C enhanced glucose-induced extracellular signal-regulated kinases 1/2 (ERK1/2) activation as shown by higher phosphorylation levels in INS-1 β-cells. Interestingly, inhibition of ERK1/2 with two structurally distinct inhibitors led to a reduction in urolithin C effect on insulin secretion. Finally, we provide data to suggest that urolithin C-mediated ERK1/2 phosphorylation involved insulin signalling in INS-1 cells. Together, these data indicate that the pharmacological action of urolithin C on insulin secretion relies, in part, on its capacity to enhance glucose-induced ERK1/2 activation. Therefore, our study extends our understanding of the pharmacological action of urolithin C in β-cells. More generally, our findings revealed that urolithin C modulated the activation of key multifunctional intracellular signalling kinases which participate in the regulation of numerous biological processes.

BACKGROUND AND PURPOSE: The pharmacology of polyphenol metabolites on beta‐cell function is largely undetermined. We sought to identify polyphenol metabolites that enhance the insulin‐secreting function of beta‐cells and to explore the underlying mechanisms. EXPERIMENTAL APPROACH: INS‐1 beta‐cells and rat isolated islets of Langerhans or perfused pancreas preparations were used for insulin secretion experiments. Molecular modelling, intracellular Ca(2+) monitoring, and whole‐cell patch‐clamp recordings were used for mechanistic studies. KEY RESULTS: Among a set of polyphenol metabolites, we found that exposure of INS‐1 beta‐cells to urolithins A and C enhanced glucose‐stimulated insulin secretion. We further characterized the activity of urolithin C and its pharmacological mechanism. Urolithin C glucose‐dependently enhanced insulin secretion in isolated islets of Langerhans and perfused pancreas preparations. In the latter, enhancement was reversible when glucose was lowered from a stimulating to a non‐stimulating concentration. Molecular modelling suggested that urolithin C could dock into the Ca(v)1.2 L‐type Ca(2+) channel. Calcium monitoring indicated that urolithin C had no effect on basal intracellular Ca(2+) but enhanced depolarization‐induced increase in intracellular Ca(2+) in INS‐1 cells and dispersed cells isolated from islets. Electrophysiology studies indicated that urolithin C dose‐dependently enhanced the L‐type Ca(2+) current for levels of depolarization above threshold and shifted its voltage‐dependent activation towards more negative potentials in INS‐1 cells. CONCLUSION AND IMPLICATIONS: Urolithin C is a glucose‐dependent activator of insulin secretion acting by facilitating L‐type Ca(2+) channel opening and Ca(2+) influx into pancreatic beta‐cells. Our work paves the way for the design of polyphenol metabolite‐inspired compounds aimed at ameliorating beta‐cell function.

International audience

International audience; The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

Urolithins are microflora human metabolites of dietary ellagic acid derivatives. There is now a growing interest in the biological activities of these compounds. Several studies suggest that urolithins have potential antioxidant, anti-inflammatory, anticancer and anti-glycative activities. Recently, our group investigated the role of urolithins as potential anti-diabetic treatments; among the four urolithins, urolithin C was the most promising compound. The purpose of this paper was to develop a rapid, sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urolithin C in rat plasma. To date, no method is reported for the quantification of urolithin C in any of the matrices. Plasma samples were extracted with ethyl acetate. Urolithin D was selected as the internal standard. The separation was carried out on a C18 Kinetex EVO column (2.1mm×150mm, 2.6μm) using a mobile phase of acetonitrile-1% aqueous formic acid solution (30:70, v/v). A triple quadrupole mass spectrometer in the negative ion mode was used for the determination of the target analyte. The monitored ion transitions were m/z 243→187 for urolithin C and m/z 259→213 for the internal standard. The calibration curve range was 4.95-1085μg/L (r2>0.994). The intra- and inter-day precisions were less than 10%; accuracies ranged from 96.6 to 109%. The mean extraction recovery of urolithins C and D was greater than 91%. No significant matrix effects and no carryover effects were observed. Small changes in LC-ESI-MS/MS conditions did not have significant effect on the determination of urolithin C. Stability tests under various conditions were also investigated. This highly specific and sensitive method was used to analyze samples collected during preclinical pharmacokinetic studies in rats. Glucuronyl and sulfate conjugates of urolithin C were the main metabolites detected in plasma.

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Background and Purpose Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca2+]i) in beta cells, in the absence of any co-stimulating factor. Experimental Approach Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca2+]i were measured using the ratiometric fluorescent Ca2+ indicator Fura-2. Ca2+ channel currents were recorded with the whole-cell patch-clamp technique. Key Results Quercetin concentration-dependently increased insulin secretion and elevated [Ca2+]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L−1), but were nearly abolished by the L-type Ca2+ channel antagonist nifedipine (1 μmol·L−1). Similar to the L-type Ca2+ channel agonist Bay K 8644, quercetin enhanced the L-type Ca2+ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca2+]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L−1), with the two drugs having cumulative effects on [Ca2+]i. Conclusions and Implications Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca2+ influx through an interaction with L-type Ca2+ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.

BACKGROUND AND PURPOSE Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic β-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting β-cells. Using the INS-1 β-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on β-cell dysfunctions induced by hydrogen peroxide (H2O2). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison.

Diabetes mellitus is a complex metabolic disorder and is considered a fast-growing global health problem. Type 2 diabetes (T2D) represents the majority of total diabetes prevalence and β-cell dysfunction has been described as a crucial point for this disease development and progression. To date, all of the common anti-hyperglycaemic drugs used for diabetes management cause undesirable side effects or problems with long-term efficacy or safety and the development of alternative approaches for the prevention as well as for the treatment of T2D might be a valuable solution to meet this rising demand. In this regards, numerous epidemiological studies indicate that exposure to certain polyphenol compounds is associated with the prevention of chronic diseases, including diabetes. Here, we review growing evidence suggesting that polyphenols can modulate the activity of various molecular targets, which are known to control β-cell function, involved in the development and the progression of this diabetes. The protective effects of polyphenols on β-cell function is reported with a particular focus on the mechanism of action behind polyphenol putative bioactivity. Animal and in vitro studies selected in this review, reporting about both flavonoid and non-flavonoid compounds, highlight the direct action of polyphenols on pancreatic β-cells, stimulating insulin secretion through the activation of specific cellular targets and protecting these cells from damages mediated by oxidative stress and inflammation, both typically elevated in diabetes. Some of the reviewed studies describe polyphenol effects comparable to those exerted by many drugs commonly used in diabetes treatment, and, in some occasions, synergistic polyphenol-drug interactions. Finally, future studies need to be addressed to the effects of specific polyphenol human and microbial metabolites, which are still poorly studied, in order to better define the preventive and therapeutic approach to contrast β-cell failure and diabetes progression.

1 The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2 To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3 Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4 We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Go6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Go6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (ɛ, δ), but not conventional or atypical PKCs. Further analyses suggest that PKCɛ is required for the activation of ERK1/2. 5 Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCɛ-dependent pathway. British Journal of Pharmacology (2006) 148, 350–365. doi:10.1038/sj.bjp.0706727

In cells overexpressing active MEKK1 to enhance c-Jun trans-activation, expression of rat cholecystokinin 1 receptor increased the activity of c-Jun while in the same experimental conditions overexpression of mouse cholecystokinin 1 receptor repressed it. This differential trans-activation is specific, since it was not observed for either the other overexpressed kinases (MEK, PKA) or for other transcription factors (ATF2, ELK-1, CREB). This differential behaviour was also detected in a human colon adenocarcinoma cell-line naturally producing high levels of endogenous MEKK1. This differential behaviour between the two receptors on the MEKK1-induced c-Jun trans-activation was independent of the activation state of JNK, of the phosphorylation level of c-Jun and of its ability to bind its specific DNA responsive elements. Two amino acids (Val43 and Phe50 in the mouse cholecystokinin 1 receptor, replaced by Leu43 and Ileu50 in the rat cholecystokinin 1 receptor) localized in the first transmembrane domain were found to play a crucial role in this differential behaviour. MEKK1 probably activates a transcriptional partner of c-Jun whose activity is maintained or increased in the presence of the rat cholecystokinin 1 receptor but repressed in the presence of the mouse cholecystokinin 1 receptor. British Journal of Pharmacology (2006) 147, 951–958. doi:10.1038/sj.bjp.0706690

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or “intramolecular chaperones” to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined α−β conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT·subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.

The aim of this study was to analyse the antisecretory mechanism of L-365,260 in vitro in isolated rabbit gastric glands. We showed that compound L-365,260, described as a non-peptide specific competitive CCK-B receptor antagonist, was able to dose-dependently inhibit [14C]-aminopyrine accumulation induced by histamine (10−4 M), carbachol (5×10−5 M), 3-isobutyl-1-methyl-xanthine (IBMX) (5×10−6 M) and forskolin (5×10−7 M) with similar IC50 values respectively of 1.1±0.6×10−7 M, 1.9±1.2×10−7 M, 4.2±2.0×10−7 M and 4.0±2.8×10−7 M. We showed that L-365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+-ATPase. We found that L-365,260 inhibited cyclic AMP-induced [14C]-aminopyrine accumulation in digitonin-permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP-dependent protein kinase (PKA) pathway. Keywords: L-365,260; isolated rabbit gastric glands; [14C]-aminopyrine accumulation; H+/K+-ATPase; protein kinase inhibitor Introduction Acetylcholine, gastrin and histamine are the three most important mediators that stimulate gastric acid secretion by interacting with specific receptors located at the basolateral membrane of the parietal cells. Acetylcholine interacts with a muscarinic M3 receptor (Kajimura et al., 1992), gastrin with a CCK-B/gastrin receptor (Kopin et al., 1992) and histamine with a histamine H2 receptor (Black et al., 1972). It has been established that the gastric histamine H2 receptor is coupled to the formation of cyclic AMP (Chew et al., 1980). Activation of the parietal cell M3 cholinoceptor or the CCK-B/gastrin receptor leads to an elevation of [Ca2+]i (Chew et al., 1992). The observation that cholinergic or gastrin stimulation of acid secretion is potentiated by histamine (Berglindh et al., 1976; Soll, 1978; Chew & Hersey, 1982; Oiry et al., 1995) suggests that cyclic AMP and [Ca2+]i interact at some level. Whatever the complexity of the mechanism of acid secretion, the binding of these ligands to the receptors on the basolateral membrane of the parietal cell generates changes in second messengers and activation of protein kinase cascades that lead to final activation of the H+/K+-adenosine triphosphatase (H+/K+-ATPase) (EC 3.6.1.36) (Sachs et al., 1976; Forte & Soll, 1989). One role of cyclic AMP is the activation of cyclic AMP-dependent protein kinase (PKA) (Chew, 1985). PKA has specific functional targets and also initiates a phosphorylation cascade through activation of other downstream protein kinases the actions of which cause proton pump activation and cytoskeletal rearrangements which are characteristic of the stimulated parietal cell. It has been suggested that Ca2+/calmodulin-dependent kinase may play an important regulatory role in the modulation of cytoskeletal structure and function. Recently, Tsunoda et al. (1992) reported that Ca2+/calmodulin-dependent kinase II (CaMK II) mediates cholinergic-stimulated parietal cell secretion. In resting parietal cells, the H+/K+-ATPase is sequestered in the cytoplasmic membrane compartments of low K+ permeability: the tubulovesicles. Stimulation of the parietal cells causes a cytoskeletal reorganization and a fusion of the tubulovesicles with the apical plasma membrane, transferring the proton pump to that surface (Forte & Soll, 1989). The H+/K+-ATPase constitutes the final step common to each secretagogue in the production of acid by parietal cells and so provides a target for inhibitors used therapeutically as anti-ulcer agents such as omeprazole (Fellenius et al., 1981). In a previous study in which we compared the acid secretion induced by the C-terminal octapeptide of cholecystokinin (CCK-8) and [Leu11]gastrin(5–17) in isolated rabbit gastric glands (Oiry et al., 1995), we confirmed the ability of histamine to potentiate the action of CCK-8 (or [Leu11]gastrin(5–17)). To better understand the cooperativity between CCK and histamine, we tested the potency of various classes of CCK receptor antagonists (L-365,260; PD-135,158; YM-022; JMV-180 and L-364,718) as inhibitors of histamine-induced [14C]-aminopyrine accumulation. We found that PD-135,158; YM-022; JMV-180 and L-364,718 had no effect on histamine-induced [14C]-aminopyrine accumulation. Surprisingly, only compound L-365,260 appeared active and even more potent than cimetidine to inhibit histamine-induced [14C]-aminopyrine accumulation. Compound L-365,260, already described in the literature as a competitive and specific CCK-B/gastrin receptor antagonist (Lotti & Chang, 1989), presented a different and specific behaviour compared to other CCK-B/gastrin antagonists (PD-135,158, YM-022 and JMV-180). It has been mentioned that in the rat in vivo, low doses of L-365,260 inhibited pentagastrin-stimulated secretion (Hirst et al., 1991), whereas higher doses inhibited basal-, histamine- and carbachol-stimulated secretion (Hirst et al., 1991; Nishida et al., 1992; Pendley et al., 1993). In order to provide a better understanding of the role of L-365,260 in acid secretion in vitro, we decided to investigate further its antisecretory mechanism in isolated rabbit gastric glands.

Insulin secretion preservation is a major issue for the prevention or treatment of type 2 diabetes. We previously showed on β-cells that quercetin (Q), but not resveratrol (R) or N-acetyl cysteine (NAC), amplified glucose-induced insulin secretion in a calcium- and ERK1/2-dependent manner. Quercetin, but not resveratrol or NAC, also protected β-cell function and hyperamplified ERK1/2 phosphorylation in oxidative stress conditions. As quercetin may interfere with other stress-activated protein kinases (JNK and p38 MAPK), we further explored MAPK cross talks and their relationships with the mechanism of the protective effect of quercetin against oxidative stress. In INS-1 insulin-secreting β-cells, using pharmacological inhibitors of MAPK pathways, we found that under oxidative stress (50 μm H2O2) and glucose-stimulating insulin secretion conditions: (i) p38 MAPK phosphorylation was increased and regulated by ERK1/2 (positively) and JNK (negatively), although p38 MAPK activation did not seem to play any significant role in oxidative stress-induced insulin secretion impairment; (ii) the JNK pathway appeared to inhibit both ERK1/2 activation and insulin secretion, although JNK phosphorylation was not significantly changed in our experimental conditions; (iii) the functionality of β-cell in the presence of oxidative stress was closely linked to the level of ERK1/2 activation, (iv) quercetin, resveratrol, or NAC inhibited H2O2 -induced p38 MAPK phosphorylation. The preservation of β-cell function against oxidative stress appears dependent on the balance between ERK1/2 and JNK activation. The protecting effect of quercetin appears due to ERK1/2 hyperactivation, possibly induced by L-type calcium channel opening as we recently showed.

International audience; Hyperglycemia is a well-known inducing factor of oxidative stress through activation of NADPH oxidase. In addition to its plasma glucose lowering effect, insulin may also have antioxidant activity and was shown to downregulate NADPH oxidase expression in vitro. In this study, we show that a short-term (3-day) intravenous insulin infusion in patients with type 2 diabetes induces normalization of both glycemia and mRNA expression of circulating monocyte p47(phox) subunit.

In the present study we compared various CCK(B) receptor antagonists and tried to detect a difference in biological activity between the C-terminal octapeptides of cholecystokinin (CCK-8) and [Leu11]gastrin-(5-17) in isolated rabbit gastric glands. Binding experiments showed that different CCK(B)/gastrin receptor agonists bound with high affinity and that antagonists inhibited this binding in accordance with a CCK(B)/gastrin pharmacological profile. [Leu11]gastrin-(5-17), CCK-8 and cionin were found to induce [14C]aminopyrine accumulation to 25% above the basal level. Under the same experimental conditions, histamine induced a response twice as great as the response obtained with [Leu11]gastrin-(5-17) or CCK-8. [Leu11]gastrin-(5-17) (10(-7) M), CCK-8 (10(-8) M) and cionin (10(-8) M) appeared to be full agonists. CCK(B)/gastrin receptor antagonists including L-365,260 (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-N-(3-methylphenyl) urea), L-364,718 (3S-(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-1H-indole-2-carboximide) (a selective CCK(A) receptor antagonist), PD-135,158 (4([2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2. 2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl] amino-4-oxo-[1S-1alpha.2beta[S*(S*)]4alpha]]-butano nate N-methyl-D-glucamine) (bicyclo system 1S-endo), YM-022 ((R)-1-[2,3-dihydro-1-(2'-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-++ +benzodiazepin-3-yl]-3-(3-methylphenyl)urea) and JMV-180 (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-O-CH2-CH2-C6H5) exhibited the same profile for inhibition of [Leu11]gastrin-(5-17) or CCK-8-induced [14C]aminopyrine accumulation in rabbit gastric glands. These results suggested that [Leu11]gastrin-(5-17) and CCK-8 induced [14C]aminopyrine accumulation by the same mechanism. [Leu11]gastrin-(5-17)- or CCK-8-induced [14C]aminopyrine accumulation was inhibited by about 40% by the histamine H2 receptor blocker cimetidine. These results are consistent with there being cooperativity between [Leu11]gastrin-(5-17) (or CCK-8) and histamine in the acid secretory pathway. Similarly, the CCK(B)/gastrin receptor antagonists were tested against histamine-induced [14C]aminopyrine accumulation and surprisingly, only compound L-365,260 appeared active and even more potent than cimetidine.

We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and cholecystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCKB receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCKB receptors were measured by inhibition of [125I]Bolton Hunter-CCK-8 (3-[125I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTP gamma S (guanosine 5'-O-(3-thio)triphosphate) or aluminum tetrafluoride (AlF4-). Activation of the G proteins by GTP gamma S or AlF4- led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCKB receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCKB receptors and their related intracellular events.

International audience; Isolated cardiomyocyte contractility was explored in a model of metabolic syndrome (the fructose-fed rat) and the effect of a treatment by a total red wine polyphenolic extract (RWPE) determined. Fructose-enriched diet impaired cardiomyocyte maximal shortening and velocities of contraction and relaxation, while RWPE was able to prevent those changes. Those studies suggest that red wine polyphenols are able to prevent the development of a cardiac phenotype commonly observed at an early stage of heart failure.

International audience; We previously showed that grape extracts enriched in different polyphenolic families were similarly able to prevent reactive oxygen species (ROS) production, although having differential effects on various features of metabolic syndrome when administered at a dose of 21 mg/kg to the fructose (60%)-fed rat (a model of metabolic syndrome). In the present work, we analyzed on the same model the effect of pure polyphenolic molecules (catechin, resveratrol, delphinidin, and gallic acid) administered at a dose of 2.1 mg/kg. Delphinidin and gallic acid prevented insulin resistance, while gallic acid prevented the elevation of blood pressure. All molecules prevented cardiac ROS overproduction and NADPH overexpression. We also showed that fructose feeding was associated with cardiac fibrosis (accumulation of collagen I) and expression of osteopontin, a factor induced by ROS and a collagen I expression inducer. Collagen I and osteopontin expressions were prevented by the administration of all polyphenolic molecules. The potential use of polyphenols in the prevention of cardiac fibrosis should be further explored.

1. The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2. To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3. Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4. We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Gö6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Gö6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (epsilon, delta), but not conventional or atypical PKCs. Further analyses suggest that PKCepsilon is required for the activation of ERK1/2. 5. Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCepsilon-dependent pathway.

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.

International audience; It is well known that the amidated C-terminal part of gastrin is crucial for its interaction with the classical seven transmembrane domain receptors CCK-1 or CCK-2. Nevertheless, over the past 10 years, several groups have characterized new binding sites using peptides related to gastrin (particularly glycine-extended forms of gastrin) on various tumoral and nontumoral cell lines. In the present study, we focused on the human astrocytic tumoral cell line U373. Although it has been described that gastrin was able to inhibit the motility of these cells, we were unable to detect any classical CCK/gastrin receptor. On the other hand, by using the radiolabeled C-terminal heptapeptide of gastrin ((125)I-G-7), we evidenced a new binding site that possessed a pharmacological profile different from the classical CCK/gastrin receptors. This new gastrin binding site seemed to be coupled to G proteins and be implicated in c-Fos transcription gene. Moreover, we showed that G-7 was able to induce a strong inhibition of U373 cell migration, a crucial biological effect when we know that astrocytoma cells' migration in brain parenchyma constitutes a major feature of malignancy in astrocytic tumors.It is well known that the amidated C-terminal part of gastrin is crucial for its interaction with the classical seven transmembrane domain receptors CCK-1 or CCK-2. Nevertheless, over the past 10 years, several groups have characterized new binding sites using peptides related to gastrin (particularly glycine-extended forms of gastrin) on various tumoral and nontumoral cell lines. In the present study, we focused on the human astrocytic tumoral cell line U373. Although it has been described that gastrin was able to inhibit the motility of these cells, we were unable to detect any classical CCK/gastrin receptor. On the other hand, by using the radiolabeled C-terminal heptapeptide of gastrin ((125)I-G-7), we evidenced a new binding site that possessed a pharmacological profile different from the classical CCK/gastrin receptors. This new gastrin binding site seemed to be coupled to G proteins and be implicated in c-Fos transcription gene. Moreover, we showed that G-7 was able to induce a strong inhibition of U373 cell migration, a crucial biological effect when we know that astrocytoma cells' migration in brain parenchyma constitutes a major feature of malignancy in astrocytic tumors.

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.

Analogues of bombesin which incorporate dipeptide or turn mimetics have been synthesized. One of them (compound 11) containing a seven-membered lactam ring revealed a good affinity for GRP/BN receptors on rat pancreatic acini (K(i) value of 1.7 +/- 0.4 nM) and on Swiss 3T3 cells (K(i) value of 1.0 +/- 0.2 nM). On the basis of this observation, antagonists containing the same dipeptide mimic were obtained by modification of the C-terminal part of the bombesin analogues. The most potent constrained compounds (15 and 17) were able to antagonize 1 nM bombesin-stimulated amylase secretion from rat pancreatic acini with high potency (K(i) = 21 +/- 3 and 3.3 +/- 1.0 nM, respectively) and 10(-7) M bombesin-stimulated ¿(3)Hthymidine incorporation into Swiss 3T3 cells (K(i) = 7.8 +/- 2. 0 and 0.5 +/- 0.1 nM, respectively).

The aim of this study was to analyze the role of cholecystokinin (CCK(B)) receptor in human lymphoblastic Jurkat T cells. We investigated the trophic effect resulting from activation of such a receptor by using the reporter gene strategy. For this purpose, we transiently transfected Jurkat T cells with the reporter plasmid p[(TRE)3-tk-Luc] and found that CCK-8 was able to dose-dependently induce luciferase expression related to activator protein-1 (AP-1) activation with a maximal response identical to that obtained with compounds known to activate AP-1 complex (quantitatively, the same level of induction was obtained with 1 nM 12-O-tetradecanoylphorbol-13-acetate, 100 microM diacylglycerol, or 4 nM epidermal growth factor). The involvement of the CCK(B) receptor in such a stimulation was demonstrated by the inhibiting effect of the selective CCK(B) receptor antagonist PD-135,158. This effect was confirmed in COS-7 cells transfected with the cDNA of CCK(B) receptor cloned from Jurkat T cells. To better understand the AP-1-dependent luciferase expression in Jurkat T cells, we tested two specific inhibitors of serine/threonine phosphatases-1 and -2A: okadaic acid and calyculin A. These compounds strongly increased the phorbol-12-myristate-13-acetate response, whereas we have not observed a contribution of phosphatase inhibitors on a CCK-8-induced luciferase activity. To confirm that CCK(B) receptors are involved in AP-1 response, we investigated the CCK-8 effect on interleukin-2 expression, a natural endogenous gene regulated by several factors, including AP-1. In Jurkat T cells activated by phorbol-12-myristate-13-acetate and phytohemagglutinin, CCK-8 induced IL-2 expression. This induction was abolished by PD-135,158. Our results indicate that CCK-8 exerts a trophic effect in Jurkat T cells through stimulation of CCK(B) receptors by modulation of expression of AP-1-regulated genes.

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1 alpha mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e. g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 mu M). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.

Introduction : Les antioxydants d’origine alimentaire pourraient prevenir la resistance a l’insuline ainsi que le developpement du stress oxydant associe aux maladies metaboliques. Parmi ceux-ci, les polyphenols tels que l’acide cafeique et ses derives (acide chlorogenique et acide chicorique) possederaient des proprietes antidiabetiques. Nous avons etudie, sur la lignee de myoblastes L6, l’influence de l’acide chicorique (AC) sur le stress oxydant, l’activite mitochondriale, la voie de l’AMPK (proteine kinase activee par l’AMP) et celle de l’insuline. Materiels et methodes: L’expression des proteines et des ARNm a ete determinee par Western Blot et qPCR. Les especes reactives de l’oxygene (ROS) ont ete quantifiees avec la sonde 2’,7-dichlorofluoresceine. La captation cellulaire de glucose a ete mesuree par l’incorporation de 2-deoxy-D-glucose[3H]. Resultats : Dans les myotubes L6, l’AC est un piegeur de ROS en condition basale et en condition de stress oxydant. L’AC augmente l’activite des systemes de defenses enzymatiques anti-oxydantes glutathion peroxydase et superoxyde dismutase (SOD). Il protege la mitochondrie contre les dommages oxydatifs en augmentant l’expression de la MnSOD. L’AC augmente l’activite du complexe II ainsi que l’expression de PGC-1α implique dans la regulation de l’expression des enzymes anti-oxydantes et la biogenese mitochondriales. L’AC stimule la voie AMPK/ACC et inhibe la voie Akt/mTOR en presence d’insuline, sans modification de la captation de glucose. Conclusion : L’AC possede des proprietes anti-oxydantes, a la fois par sa capacite a neutraliser les ROS et a augmenter les systemes enzymatiques de defense anti-oxydante. L’AC stimule egalement la biogenese mitochondriale et protege les mitochondries contre les dommages oxydatifs, tout en ameliorant leur capacite a oxyder les acides gras en stimulant l’AMPK et en augmentant l’activite du complexe II. Ces resultats suggerent que le potentiel de l’acide chicorique a prevenir ou traiter les pathologies associees au syndrome metabolique merite d’etre etudie de facon plus approfondie.

We have investigated the potential role of gastrin in the regulation of cell growth in human astrocytic tumors. To this end we have used synthetic analogs of gastrin and cholecystokinin (CCK) which behave as gastrin and/or CCK antagonists, e.g. compounds JMV-97, JMV-209 and JMV-179. Their effects on astrocytic tumor cell proliferation was investigated by the use of the colorimetric MTT assay. The in vitro biological models used in the present study included the SW1088, U87 and U373 astrocytic tumor cell lines. The results demonstrated marked influence of gastrin and CCK antagonists in the regulation of astrocytic tumor growth. This suggests that gastrin and/or CCK antagonists might be tested in experimental glioblastoma.

Introduction Dans la lignee beta INS-1, nous avons precedemment montre que la quercetine, un flavonoide, potentialise la secretion d’insuline et la phosphorylation de ERK1/2 induites par le glucose ou le glibenclamide. Ces derniers induisent la fermeture des canaux KATP, une depolarisation membranaire suivie de l’ouverture des canaux calciques de type L et une entree de calcium provoquant la secretion d’insuline. Nous nous sommes interesses a l’effet de la quercetine sur la concentration de calcium intracellulaire ([Ca]i) apres depolarisation membranaire des cellules INS-1. Pour cela, nous avons evalue les effets de la nifedipine (antagoniste des canaux calciques de type L) ou de la thapsigargine (inhibiteur de la Ca2+ ATPase du reticulum endoplasmique (SERCA) sur la potentialisation de la secretion d’insuline et sur les variations de [Ca2+]i induites par la quercetine. Materiels et methodes La secretion d’insuline et l’activation de ERK1/2 ont ete evaluees apres 60 minutes d’incubation, respectivement par transfert de fluorescence (HTRF) et par Western blot. La [Ca]i a ete determinee par imagerie calcique avec la sonde fluorescente fura-2. Resultats La quercetine (20 μM) induit une secretion d’insuline mineure (× 1,3), active ERK1/2 (× 2,5) et provoque une faible augmentation de [Ca]i. Elle potentialise l’augmentation de [Ca]i (× 1,6) induite par depolarisation (15 mM KCl) et la secretion d’insuline (× 2) gluco- ou KCl-dependante. La nifedipine (1 μM) inhibe l’effet potentialisateur de la quercetine sur l’augmentation de [Ca2 +]i et sur la secretion d’insuline. Par contre, la thapsigargine (1 μM) ne modifie pas l’effet potentialisateur de la quercetine. Conclusion Nos resultats montrent un effet direct de la quercetine sur l’augmentation de [Ca]i dans la cellule beta impliquant des canaux calciques de type L et non les SERCA. La quercetine, par une augmentation de [Ca]i basale conduisant a une activation de ERK1/2, sensibiliserait la cellule beta aux divers stimulants et ainsi amplifierait leur reponse secretoire.

Introduction Nous avons montre que le syndrome metabolique (SM), induit experimentalement par une alimentation enrichie en fructose 60% (F), etait associe a une augmentation de la pression arterielle, une fibrose cardiaque et une hyperproduction d'especes oxygenees reactives, et que ces manifestations etaient prevenues par l'administration d'un extrait polyphenolique total de vin (EPV, 21mg/kg/jour) ou de polyphenols purs (2mg/kg/jour). Cependant, aucune alteration fonctionnelle ou morphologique n'a ete retrouvee in vivo par echocardiographie. Toutefois, nos donnees recentes indiquent que la contractilite des cardiomyocytes isoles peut etre alteree, meme en absence de manifestations echocardiographiques (Andre et al., 2010). Nous avons donc determine les parametres-cles du couplage excitation/contraction de cardiomyocytes isoles apres 2, 8 et 13 semaines de regime F, ainsi que les effets preventifs eventuels de l'EPV. Materiels et methodes La fraction de raccourcissement cellulaire (FRC) et les modifications du Ca2 + cytosolique (Indo-1) des cardiomyocytes ont ete determinees simultanement sous stimulations en champ (0,5 Hz, 22°C, 1,8mM Ca 2+ ). Resultats Chez les animaux temoins (C), la FRC ete similaire a 2 et 8 semaines, avant de decroitre spontanement a 16 semaines. Chez les animaux F, la FRC a ete significativement diminuee par rapport au groupe C a 2, 8 et 13 semaines, la difference entre C et F etant maximale a 8 semaines. De plus, chez les rats F, les vitesses de contraction et de relaxation ont ete diminuees et le re-captage du Ca2 + cytosolique ralenti (augmentation de Tau), suggerant une alteration des pompes Serca2a. Chez les rats C, l'EPV diminue la vitesse de relaxation et le recaptage calcique a 8 semaines. Chez les rats F, l'EPV previent l'alteration du FRC et le ralentissement de contraction et de relaxation, tout en accentuant le ralentissement du recaptage calcique. Conclusion La contractilite et la cinetique calcique des cardiomyocytes sont alterees precocement par un regime inducteur du SM. Un EPV anti-oxydant previent l'alteration de contractilite.

Objectif La quercetine, un polyphenol de la famille des flavonoides, possede in vivo des proprietes antidiabetiques non explorees directement sur les cellules β. Dans la lignee β INS-1, les effets de la quercetine ont ete evalues sur la secretion d'insuline induite par le glucose (ou le glibenclamide) et ce parallelement a l'activation de ERK1/2, une voie impliquee dans la fonctionnalite et la survie des cellules β. Les effets de la quercetine ont egalement ete etudies sur le dysfonctionnement des cellules β induit par le peroxyde d'hydrogene (H 2 O 2 ). Les effets de la N-acetyl-L-cysteine (NAC) et du resveratrol (polyphenol de type stilbene), deux antioxydants possedant des proprietes antidiabetiques, ont ete compares a ceux de la quercetine. Materiels et Methodes La viabilite, la secretion d'insuline et l'activation de ERK1/2 ont ete evaluees apres 60 minutes d'incubation, respectivement par la technique du MTT, par transfert de fluorescence (HTRF) et par Western blot. Resultats En absence de glucose, la quercetine (20 μM) induit une secretion d'insuline mineure (x1,3) et active ERK1/2 (x2,5). En presence de 8,3 mM glucose (ou de 0,01 μM glibenclamide) la quercetine potentialise la secretion d'insuline (x2,5) et la phosphorylation de ERK1/2 (x4). Les etudes cinetiques et l'utilisation d'inhibiteurs pharmacologiques montrent que la voie ERK1/2 (mais non la voie de la PKA) joue un role majeur dans ce phenomene de potentialisation. En presence de 50 μM H 2 O 2 , la fonctionnalite et la viabilite des cellules β sont diminuees d'environ 50 %. La quercetine (20 μM) previent totalement ces alterations et entraine une sur-activation de ERK1/2 (x6 vs glucose). Le resveratrol ou la NAC ne presentent aucun effet sur ces differents parametres. Conclusion En sur-activant la voie ERK1/2, la quercetine potentialise la secretion d'insuline gluco-dependante et protege la viabilite et la fonctionnalite des cellules β soumises a un stress oxydant. Sachant que le diabete de type 2 est lie a une alteration de la fonction cellulaire β associee a une augmentation du stress oxydant, la quercetine, par un mecanisme d'action original, pourrait ouvrir la voie a une therapeutique preventive innovante.

Introduction L’hyperglycemie chronique induit un stress oxydant implique dans la pathogenie des complications vasculaires du diabete. Nos objectifs etaient d’analyser, chez des patients diabetiques de type 2 (DT2) mal controles sous antidiabetiques oraux (ADO), l’expression d’une enzyme cle du stress oxydant, la NADPH oxydase (NADPHox), et sa regulation apres correction rapide de l’hyperglycemie. Materiels et Methodes L’expression des transcrits de deux sous-unites de la NADPHox, p22 phox (membranaire) et p47 phox (cytosolique), a ete etudiee par Q-PCR (Lightcycler-Roche) dans des monocytes isoles de patients non diabetiques (ND, n = 6), DT2 controles sous ADO (C, n = 6) et DT2 non controles sous ADO (NC, n = 12). Dans ce dernier groupe, l’expression a ete evaluee avant (J0) et apres trois jours (J3) d’insulinotherapie IV normalisant la glycemie. L’expression des transcrits a ete normalisee par la β-2-microglobuline. Resultats Les patients des groupes C et NC sont âges de 56,4 ± 7,9 et 64,0 ± 4,5 ans, ont un DT2 connu depuis 8,3 ± 2,9 et 9,1 ± 3,3 ans et presentent une HbA1c de 7,1 ± 0,4 % et 10,26 ± 1,0 %, respectivement. Dans les monocytes, les niveaux d’expression de la p47 phox sont : 2,14 ± 0,83 (ND) ; 2,57 ± 1,16 (C) ; 3,52 ± 1,86 (NC, J0). Nous observons une tendance a l’augmentation non significative de l’expression de la p47 phox dans le groupe NC a J0 vs . ND et C. En revanche, pour la p22 phox , aucune difference d’expression n’est constatee entre les groupes. Dans le groupe NC, apres normalisation des glycemies (J3), nous observons une diminution significative de l’expression de la p47 phox : 2,57 ± 1,58 vs . 3,52 ± 1,86 (p phox . Conclusion Nos resultats montrent une tendance a l’augmentation de l’expression de la p47 phox de la NADPHox chez des patients DT2 en hyperglycemie chronique et une diminution significative de cette expression lors de la correction rapide de l’hyperglycemie par une insulinotherapie IV. Ces variations d’expression, a confirmer dans des populations plus importantes, sont en faveur de la gluco-dependance du stress oxydant dans le DT2, et suggerent la possibilite de sa suppression rapide lors de la correction glycemique par l’insuline.

Introduction Le stress oxydant joue un role preponderant dans l’apparition du diabete de type 2. La cellule beta pancreatique est particulierement sensible au stress oxydant car elle n’exprime que faiblement les enzymes de detoxification. Ainsi, l’augmentation des ROS pourrait constituer un facteur inducteur d’alterations precoces dans cette cellule. Par consequent, le traitement par des molecules anti-oxydantes pourrait proteger la cellule beta des agressions oxydantes et constituerait ainsi une nouvelle approche pharmacologique dans la prevention du diabete de type 2. L’objectif de notre travail a ete d’etudier les effets protecteurs de deux molecules polyphenoliques de familles differentes, la Quercetine et le Resveratrol, sur la viabilite et la fonctionnalite de la cellule beta, lors d’un stress oxydant induit de facon exogene. Materiels et methodes Des cellules INS-1 insulinosecretrices ont ete incubees en presence d’H2O2 avec ou sans N-acetyl cysteine (NAC), Quercetine ou Resveratrol. La viabilite et la fonctionnalite ont ete evaluees apres 60 minutes d’incubation par la technique du MTT et la mesure de secretion d’insuline induite par le glucose (8,3 mM). La phosphorylation des proteines kinases ERK1/2, impliquees dans la fonctionnalite de la cellule beta, a ete determinee par Western Blot apres 5 mn d’incubation. Resultats H2O2 inhibe la viabilite cellulaire et la secretion d’insuline induite par le glucose de facon dose-dependante (CI50≈50 μm). La Quercetine (20 μM) previent totalement l’alteration de la viabilite et partiellement l’alteration de la fonctionnalite induites par 50 μM H2O2, alors que le Resveratrol (20 μM) et la NAC (0,1 mM) sont sans effet. La phosphorylation de ERK1/2, apres traitement par 0,5 mM H2O2, est fortement diminuee par l’inhibiteur specifique de la voie ERK1/2 (U0126). La Quercetine (20 μM) diminue la phosphorylation de ERK1/2 alors que le Resveratrol (20 μM) est sans effet. Conclusion Un stress oxydant exogene altere la viabilite et la fonctionnalite de la cellule beta pancreatique. Contrairement au Resveratrol (stilbene), la Quercetine (flavonoide) previent ces alterations. Les effets de la Quercetine s’accompagnent d’une modulation de la voie ERK1/2.

The objective of our thesis was to study the anti-diabetic potential of metabolites of ellagic acid tanins, present notably in pomegranate and nuts, that are formed by the colon microbiote. The metabolites are urolithins A, B, C and D.The first part of thesis is bibliographic and reviews: •The control of glycemic plasma levels, and in particular the role of insulin secretion in this process; •The pathophysiology of Type 2 Diabetes (T2D); •The various polyphenols and their metabolites, along with their potential anti-diabetic activity.The second part describes the effects of urolithins on various experimental models: •On a model of insulin secreting beta cells (the INS-1cell line), urolithins concentration-dependently amplified insulin secretion induced by glucose, but also by insulinotropic drugs used in the treatment of T2D such as a GLP-1 analogue or a sulfonylurea. In addition, urolithins were able to induce insulin secretion on cells rendered unresponsive to glucose by oxidative stress. •The insulinotropic effect of urolithins was also confirmed on isolated rat islets of Langerhans. •As urolithin C appeared to be the most promising antidiabetic compound, we further characterized its activity on an ex vivo model mimicking the physiological situation, the isolated infused pancreas. While urolithin C (20µM) had no effect in the presence of 5 mM glucose concentration, it amplified the stimulation of insulin secretion in the presence of 8.3mM glucose. The effect of urolithin C was also strictly glucose-dependent, as insulin secretion immediately returned to basal level when glucose concentration was switched from 8.3 to 5mM glucose in the presence of urolithin C. •We also conducted studies aiming at designing a validated methodology for rat plasma urolithin C determination using a liquid chromatography-electrospray ionization-tandem mass spectrometry method. The applicability of this assay was demonstrated in a preclinical pharmacokinetic study carried out in rats receiving intraperitoneal administration of urolithin C (10mg/kg). We found that the urolithin C followed a three-compartment model, suggesting a long-term tissue storage of urolithin C.Some other (confidential) results, not described in this abstract, confirmed urolithin C as a potential glucose-dependent insulinotropic treatment for type 2 diabetes.; Notre travail de thèse avait pour objet l’étude du potentiel anti-diabétique des urolithines A, B, C et D, métabolites de polyphénols formés par le microbiote colique à partir des tanins de l’acide ellagique (présents notamment dans la grenade et les noix). La première partie, bibliographique, constitue un rappel :•de la régulation de l’équilibre glycémique et le rôle de la sécrétion d’insuline dans cette régulation ; •de l ‘épidémiologie et la physiopathologie du diabète type 2 (DT2) ; •des polyphénols et leurs métabolites, ainsi que de leurs effets antidiabétiques potentiels.La seconde partie décrit les effets des urolithines sur différents modèles expérimentaux : •Sur un modèle de cellules β insulino-sécrétrices (lignée INS-1), les urolithines induisent une amplification concentration-dépendante de la sécrétion d’insuline induite par le glucose, mais également par d’autres sécrétagogues comme un analogue du GLP-1 ou une sulfonylurée (médicaments utilisés dans le diabète). Les urolithines préviennent également l’altération sécrétoire induite par un stress oxydant. •L’effet insulino-sécrétoire des urolithines a été confirmé sur îlots de Langerhans isolés. •L’urolithine C étant apparu comme le composé le plus prometteur, nous avons poursuivi la caractérisation de son activité sur un modèle ex vivo mimant la situation physiologique, le pancréas isolé perfusé. Alors que l’effet sécrétoire de l’urolithine C n’apparaît pas en présence de 5mM de glucose, l’urolithine C (20µM) a stimulé la sécrétion d’insuline dans des conditions de stimulation modérée de la sécrétion d’insuline par le glucose (8.3mM). Cet effet est strictement dépendant du glucose, la sécrétion d’insuline retournant immédiatement à son niveau basal lors du passage de 8,3 à 5mM de glucose en présence d’urolithine C. •Des études de pharmacocinétique ont permis de mettre au point une méthodologie de dosage plasmatique de l’urolithine C dans le plasma de rat par chromatographie liquide / ionisation electrospray /spectrographie de masse en tandem. Cette méthodologie a été appliquée à une première étude pharmacocinétique chez le rat après injection de 10mg/kg d’urolithine C par voie intra-péritonéale. Cette étude montre notamment que le profil pharmacocinétique suit un modèle à 3 compartiments et suggère un stockage tissulaire du composé.D’autres résultats (confidentiels) ne peuvent être évoqués dans ce résumé mais confirment l’intérêt potentiel de l’urolithine C dans le traitement du diabète de type 2 en tant que médicament insulinotrope dépendant du glucose.

The objective of our thesis was to study the anti-diabetic potential of metabolites of ellagic acid tanins, present notably in pomegranate and nuts, that are formed by the colon microbiote. The metabolites are urolithins A, B, C and D.The first part of thesis is bibliographic and reviews: •The control of glycemic plasma levels, and in particular the role of insulin secretion in this process; •The pathophysiology of Type 2 Diabetes (T2D); •The various polyphenols and their metabolites, along with their potential anti-diabetic activity.The second part describes the effects of urolithins on various experimental models: •On a model of insulin secreting beta cells (the INS-1cell line), urolithins concentration-dependently amplified insulin secretion induced by glucose, but also by insulinotropic drugs used in the treatment of T2D such as a GLP-1 analogue or a sulfonylurea. In addition, urolithins were able to induce insulin secretion on cells rendered unresponsive to glucose by oxidative stress. •The insulinotropic effect of urolithins was also confirmed on isolated rat islets of Langerhans. •As urolithin C appeared to be the most promising antidiabetic compound, we further characterized its activity on an ex vivo model mimicking the physiological situation, the isolated infused pancreas. While urolithin C (20µM) had no effect in the presence of 5 mM glucose concentration, it amplified the stimulation of insulin secretion in the presence of 8.3mM glucose. The effect of urolithin C was also strictly glucose-dependent, as insulin secretion immediately returned to basal level when glucose concentration was switched from 8.3 to 5mM glucose in the presence of urolithin C. •We also conducted studies aiming at designing a validated methodology for rat plasma urolithin C determination using a liquid chromatography-electrospray ionization-tandem mass spectrometry method. The applicability of this assay was demonstrated in a preclinical pharmacokinetic study carried out in rats receiving intraperitoneal administration of urolithin C (10mg/kg). We found that the urolithin C followed a three-compartment model, suggesting a long-term tissue storage of urolithin C.Some other (confidential) results, not described in this abstract, confirmed urolithin C as a potential glucose-dependent insulinotropic treatment for type 2 diabetes., Notre travail de thèse avait pour objet l’étude du potentiel anti-diabétique des urolithines A, B, C et D, métabolites de polyphénols formés par le microbiote colique à partir des tanins de l’acide ellagique (présents notamment dans la grenade et les noix). La première partie, bibliographique, constitue un rappel :•de la régulation de l’équilibre glycémique et le rôle de la sécrétion d’insuline dans cette régulation ; •de l ‘épidémiologie et la physiopathologie du diabète type 2 (DT2) ; •des polyphénols et leurs métabolites, ainsi que de leurs effets antidiabétiques potentiels.La seconde partie décrit les effets des urolithines sur différents modèles expérimentaux : •Sur un modèle de cellules β insulino-sécrétrices (lignée INS-1), les urolithines induisent une amplification concentration-dépendante de la sécrétion d’insuline induite par le glucose, mais également par d’autres sécrétagogues comme un analogue du GLP-1 ou une sulfonylurée (médicaments utilisés dans le diabète). Les urolithines préviennent également l’altération sécrétoire induite par un stress oxydant. •L’effet insulino-sécrétoire des urolithines a été confirmé sur îlots de Langerhans isolés. •L’urolithine C étant apparu comme le composé le plus prometteur, nous avons poursuivi la caractérisation de son activité sur un modèle ex vivo mimant la situation physiologique, le pancréas isolé perfusé. Alors que l’effet sécrétoire de l’urolithine C n’apparaît pas en présence de 5mM de glucose, l’urolithine C (20µM) a stimulé la sécrétion d’insuline dans des conditions de stimulation modérée de la sécrétion d’insuline par le glucose (8.3mM). Cet effet est strictement dépendant du glucose, la sécrétion d’insuline retournant immédiatement à son niveau basal lors du passage de 8,3 à 5mM de glucose en présence d’urolithine C. •Des études de pharmacocinétique ont permis de mettre au point une méthodologie de dosage plasmatique de l’urolithine C dans le plasma de rat par chromatographie liquide / ionisation electrospray /spectrographie de masse en tandem. Cette méthodologie a été appliquée à une première étude pharmacocinétique chez le rat après injection de 10mg/kg d’urolithine C par voie intra-péritonéale. Cette étude montre notamment que le profil pharmacocinétique suit un modèle à 3 compartiments et suggère un stockage tissulaire du composé.D’autres résultats (confidentiels) ne peuvent être évoqués dans ce résumé mais confirment l’intérêt potentiel de l’urolithine C dans le traitement du diabète de type 2 en tant que médicament insulinotrope dépendant du glucose.