Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis.

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.

Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human.

Barbiturate infusion for intractable intracranial hypertension and its effect on brain oxygenation.

OBJECTIVE: Barbiturate-induced coma can be used in patients to treat intractable intracranial hypertension when other therapies, such as osmotic therapy and sedation, have failed. Despite control of intracranial pressure, cerebral infarction may still occur in some patients, and the effect of barbiturates on outcome remains uncertain. In this study, we examined the relationship between barbiturate infusion and brain tissue oxygen (PbtO2). METHODS: Ten volume-resuscitated brain-injured patients who were treated with pentobarbital infusion for intracranial hypertension and underwent PbtO2 monitoring were studied in a neurosurgical intensive care unit at a university-based Level I trauma center. PbtO2, intracranial pressure (ICP), mean arterial pressure, cerebral perfusion pressure (CPP), and brain temperature were continuously monitored and compared in settings in which barbiturates were or were not administered. RESULTS: Data were available from 1595 hours of PbtO2 monitoring. When pentobarbital administration began, the mean ICP, CPP, and PbtO2 were 18 +/- 10, 72 +/- 18, and 28 +/- 12 mm Hg, respectively. During the 3 hours before barbiturate infusion, the maximum ICP was 24 +/- 13 mm Hg and the minimum CPP was 65 +/- 20 mm Hg. In the majority of patients (70%), we observed an increase in PbtO2 associated with pentobarbital infusion. Within this group, logistic regression analysis demonstrated that a higher likelihood of compromised brain oxygen (PbtO2 < 20 mm Hg) was associated with a decrease in pentobarbital dose after controlling for ICP and other physiological parameters (P < 0.001). In the remaining 3 patients, pentobarbital was associated with lower PbtO2 levels. These patients had higher ICP, lower CPP, and later initiation of barbiturates compared with patients whose PbtO2 increased. CONCLUSION: Our preliminary findings suggest that pentobarbital administered for intractable intracranial hypertension is associated with a significant and independent increase in PbtO2 in the majority of patients. However, in some patients with more compromised brain physiology, pentobarbital may have a negative effect on PbtO2, particularly if administered late. Larger studies are needed to examine the relationship between barbiturates and cerebral oxygenation in brain-injured patients with refractory intracranial hypertension and to determine whether PbtO2 responses can help guide therapy.

Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection.

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.

TCRgamma silencing during alphabeta T cell development depends upon pre-TCR-induced proliferation.

During thymus development, immature T cells become committed to two distinct lineages based upon expression of alphabeta or gammadelta TCR. In the alphabeta lineage, developing thymocytes progressively extinguish transcription of the TCRgamma genes by a poorly understood process known as gamma silencing. We show that alphabeta lineage thymocytes in mice lacking a functional pre-TCR undergo limited proliferation and fail to silence TCRgamma genes during development. Stimulation of pre-TCR-deficient immature thymocytes with anti-CD3 Abs does not directly down-regulate TCRgamma transcription but restores TCRgamma silencing following proliferation. Collectively our data reveal an important role for pre-TCR induced proliferation in activating the TCRgamma silencer in alphabeta lineage thymocytes, a process that may reinforce alphabeta or gammadelta lineage commitment.

Differential sensitivity of GLUT1- and GLUT2-expressing beta cells to streptozotocin.

Streptozotocin injection in animals destroys pancreatic beta cells, leading to insulinopenic diabetes. Here, we evaluated the toxic effect of streptozotocin (STZ) in GLUT2(-/-) mice reexpressing either GLUT1 or GLUT2 in their beta cells under the rat insulin promoter (RIPG1 x G2(-/-) and RIPG2 x G2(-/-) mice, respectively). We demonstrated that injection of STZ into RIPG2 x G2(-/-) mice induced hyperglycemia (&gt;20 mM) and an approximately 80% reduction in pancreatic insulin content. In vitro, the viability of RIPG2 x G2(-/-) islets was also strongly reduced. In contrast, STZ did not induce hyperglycemia in RIPG1 x G2(-/-) mice and did not reduce pancreatic insulin content. The viability of in vitro cultured RIPG1 x G2(-/-) islets was also unaffected by STZ. As islets from each type of transgenic mice were functionally indistinguishable, these data strongly support the notion that STZ toxicity toward beta cells depends on the expression of GLUT2.

The weaker points of fish acute toxicity tests and how tests on embryos can solve some issues.

Fish acute toxicity tests play an important role in environmental risk assessment and hazard classification because they allow for first estimates of the relative toxicity of various chemicals in various species. However, such tests need to be carefully interpreted. Here we shortly summarize the main issues which are linked to the genetics and the condition of the test animals, the standardized test situations, the uncertainty about whether a given test species can be seen as representative to a given fish fauna, the often missing knowledge about possible interaction effects, especially with micropathogens, and statistical problems like small sample sizes and, in some cases, pseudoreplication. We suggest that multi-factorial embryo tests on ecologically relevant species solve many of these issues, and we shortly explain how such tests could be done to avoid the weaker points of fish acute toxicity tests.

Urocortins improve dystrophic skeletal muscle structure and function through both PKA- and Epac-dependent pathways.

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Peroxisome proliferator-activated receptor (PPAR)-beta as a target for wound healing drugs: what is possible?

Peroxisome proliferator-activated receptor (PPAR) dysfunction has been implicated in the manifestation of many diseases and illnesses, ranging from obesity to cancer. Herein, we discuss the role of PPARbeta, one of the three PPAR isotypes, during wound healing. While PPARbeta expression is undetectable in unchallenged and healthy adult interfollicular mouse skin, it is robustly re-activated in stress situations, such as upon phorbol ester treatment, hair plucking and cutaneous wounding. The inflammatory reaction associated with a skin injury activates the keratinocytes at the edges of the wound. This activation involves PPARbeta, whose expression and activity as transcription factor are up-regulated by pro-inflammatory signals. The re-activation of PPARbeta influences three important properties of the activated keratinocytes that are vital for rapid wound closure, namely, survival, migration and differentiation. The anti-apoptotic and, thus, survival role of PPARbeta is mediated by the up-regulation of expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1. Both kinases are required for the full activation of the Akt1 survival cascade. Therefore, the up-regulation of PPARbeta, early after injury, appears to be important to maintain a sufficient number of viable keratinocytes at the wound edge. At a later stage of wound repair, the stimulation of keratinocyte migration and differentiation by PPARbeta is also likely to be important for the formation of a new epidermis at the wounded area. Consistent with these observations, the entire wound healing process is delayed in PPARbeta +/- mice and wound closure is retarded by 2-3 days. The multiple roles of PPARbeta in the complex keratinocyte response after injury and during skin repair certainly justify a further exploration of its potential as a target for wound healing drugs.

Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.

By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.

FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemotherapeutic drugs, and gamma irradiation.

FLICE-inhibitory protein, FLIP (Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT), which contains two death effector domains and an inactive caspase domain, binds to FADD and caspase-8, and thereby inhibits death receptor-mediated apoptosis. Here, we characterize the inhibitory effect of FLIP on a variety of apoptotic pathways. Human Jurkat T cells undergoing Fas ligand-mediated apoptosis in response to CD3 activation were completely resistant when transfected with FLIP. In contrast, the presence of FLIP did not affect apoptosis induced by granzyme B in combination with adenovirus or perforin. Moreover, the Fas ligand, but not the perforin/granzyme B-dependent lytic pathway of CTL, was inhibited by FLIP. Apoptosis mediated by chemotherapeutic drugs (i.e., doxorubicin, etoposide, and vincristine) and gamma irradiation was not affected by FLIP or the absence of Fas, indicating that these treatments can induce cell death in a Fas-independent and FLIP-insensitive manner.

Torasemide is an effective diuretic in the newborn rabbit.

The effect of intravenous (i.v.) torasemide on diuresis and renal function was evaluated in three groups of normoxemic, 5- to 10-day-old, newborn New Zealand White rabbits. The animals of group 1 received 0.2 mg/kg of torasemide i.v., whereas in group 2 an i.v. dose of 1.0 mg/kg was given. The third group of animals received a bolus i.v. dose of 1.0 mg/kg torasemide with continuous i.v. replacement of estimated urinary fluid and electrolyte losses. Torasemide proved to be an effective, potassium-sparing diuretic, without significant effect on glomerular filtration rate (GFR). Renal blood flow (RBF) fell and the renal vascular resistance (RVR) rose in all three groups of animals, although the rise in RVR in group 3 was not significant. These changes in renal hemodynamics were most pronounced in the animals of group 2 and are probably secondary to torasemide-induced hypovolemia (2.8% loss of body weight) and accompanying humoral reactions, such as an increase in angiotensin II (not measured). When the latter is prevented by simultaneous re-infusion of an electrolyte solution (group 3), replacing urinary losses, GFR increases and the changes in RBF and RVR are blunted. We conclude that torasemide is an effective, potassium-sparing diuretic in newborn rabbits. No evidence was found for a vasodilatory action of the drug.

Defining new criteria for selection of cell-based intestinal models using publicly available databases.

BACKGROUND: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. RESULTS: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. CONCLUSIONS: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis.

Cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is overexpressed in many cancers. Inhibition of COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer development in humans and suppresses tumor growth in animal models. The anti-cancer effect of NSAIDs seems to involve suppression of tumor angiogenesis, but the underlying mechanism is not completely understood. Integrin alpha V beta 3 is an adhesion receptor critically involved in mediating tumor angiogenesis. Here we show that inhibition of endothelial-cell COX-2 by NSAIDs suppresses alpha V beta 3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of fibroblast growth factor-2-induced angiogenesis in vivo. These results establish a novel functional link between COX-2, integrin alpha V beta 3 and Cdc42-/Rac-dependent endothelial-cell migration. Moreover, they provide a rationale to the understanding of the anti-angiogenic activity of NSAIDs.