Transepithelial Transport of Macromolecular Substances in IL-4 Treated Human Intestinal T84 Cell Monolayers

The effect of interleukin-4 (IL-4), a cytokine associated with allergy and inflammation, on the permeability of the intestinal epithelium was investigated. IL-4 reduced transepithelial electrical resistance (TER) and increased permeation to horseradish peroxidase (HRP) and Lucifer Yellow (LY) of human intestinal T84 cell monolayers. The increased permeation due to IL-4 treatment was also observed at 4 °C. The permeability of T84 cell monolayers to β-lactogulobulin (β-Lg), ovalbumin (OVA), and fluorescein isothiocyanate (FITC)-dextran of various molecular sizes was also high in the IL-4-treated cell monolayers. Sodium azide (NaN₃), which inhibits ATP synthesis of the cells, did not inhibit the increases in these substances. Even 150 kDa FITC-dextran significantly permeated the T84 cells when the monolayers were treated with IL-4. These results suggest that fairly large molecules are able to permeate intestinal epithelial monolayers via the energy-independent paracellular pathway when the monolayers are exposed to excessive IL-4.     

Permeation of volatile compounds through starch films

The aim of this study was to gain insight into the factors that affect the permeation of volatiles through starch films. These films were obtained by casting gelatinized starch/water/glycerol mixtures. The films were dried and conditioned under different conditions (temperature and relative humidity) resulting in films that vary in the degree of starch crystallinity and glycerol and water content. The permeation of two model volatiles (carvone and diacetyl) at 20 degrees C and at 30, 60, or 90% relative humidity (RH) was analyzed gravimetrically. Further, the solubility of the two model compounds (under conditions where the permeation experiments were carried out) was determined. From the obtained permeation and solubility data, the diffusion coefficients of these compounds in the different starch films were calculated. The crystallinity in the starch films increased with increasing water content of the films during preparation. The water content of the resulting films in turn increased with increasing glycerol and when the films were exposed to a higher RH during drying or conditioning. For films with the same composition, the flux for diacetyl was greater than for carvone. The solubilities of diacetyl and carvone were slightly dependent on the properties of the films. It was found that with increasing starch crystallinity the diffusion coefficient for both compounds decreases, which is probably due to the impermeability of starch crystallites. Interestingly, in films with about the same extent of crystallinity, the diffusion can be described with the free volume model, with water and glycerol determining the amount of free volume.     

Fertility and Developmental Toxicity Assessment in Rats and Rabbits with LY500307, a Selective Estrogen Receptor Beta (ERβ) Agonist

LY500307 is a selective estrogen receptor beta (ERβ) agonist that was developed for the treatment of benign prostatic hyperplasia. The in vitro functional selectivity of LY500307 for ERβ agonist activity is 32‐fold above the activity at the alpha receptor (ERα). LY500307 was evaluated in a series of male (M) and female (F) rat fertility and rat and rabbit embryo‐fetal development (EFD) studies, using 20 or 25 animals/group. LY500307 was administered daily by oral gavage starting 2 weeks (F) or 10 weeks (M) before mating, during cohabitation, until necropsy (M) or through gestation day (GD) 6 (F) in the fertility studies and from GD 6 to 17 (rats) or GD 7 to 19 (rabbits) in the EFD studies. Dosage levels of LY500307 ranged from 0.03 to 10 mg/kg/day for rats and from 1 to 25 mg/kg/day for rabbits. Fertility, estrous, maternal reproductive endpoints, conceptus viability, sperm parameters, organ weights, and histopathology were evaluated in the fertility studies. Maternal reproductive endpoints and fetal viability, weight, and morphology were evaluated in the EFD studies. Toxicokinetics were assessed in satellite animals. At 10 mg/kg/day in the male fertility study, findings included decreased body weight (BW); food consumption (FC); fertility, mating, and conception indices; sperm concentration; and reproductive tissue weight (associated with atrophic histologic changes). In the female fertility study, effects included decreased BW and FC at ≥0.3 mg/kg/day and persistent diestrus, delayed mating, and reduced fertility/conception indices at 3 mg/kg/day. In the rat EFD study, findings included decreased maternal BW and FC and increased incidences of adverse clinical signs, abortion, maternal mortality/moribundity, postimplantation loss, and fetal skeletal variations at 3 mg/kg/day. Effects in the rabbit EFD study were limited to decreases in maternal BW and FC at 25 mg/kg/day. In general, systemic maternal exposure increased proportionally with dosage in rats, but less than proportionally in rabbits. In conclusion, the no‐observed adverse effect levels following LY500307 administration were 1 mg/kg/day for male rat fertility, 0.3 mg/kg/day for female rat fertility and EFD, and 25 mg/kg/day for rabbit EFD. Adverse reproductive and developmental effects only occurred at or above parentally toxic dosage levels and were considered predominantly due to off‐target ERα effects.     

Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 microM) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl)amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 micromol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility.     

Transepithelial transport of p-coumaric acid and gallic acid in Caco-2 cell monolayers

The transepithelial transport of such common dietary phenolic acids as p-coumaric acid (CA) and gallic acid (GA) across Caco-2 cell monolayers was examined. CA transport was dependent on pH, and in a vectorial manner in the apical-basolateral direction. The permeation was concentration-dependent and saturable, the Michaelis constant and maximum velocity being 17.5 mM and 82.7 nmol min(-1) (mg of protein)(-1), respectively. Benzoic acid and acetic acid inhibited the permeation of CA. These results indicate that the transepithelial transport of CA was via the monocarboxylic acid transporter (MCT). On the other hand, the permeation of GA was not in a polarized manner, was independent of pH and linearly increased with increasing concentration of GA. The transport rate of GA was about 100 times lower than that of CA, suggesting the transepithelial transport of GA to be via the paracellular pathway. Dietary phenolic acids thus showed diversified characteristics in their intestinal absorption.     

Effects of Food Lectins on the Transport System of Human Intestinal Caco-2 Cell Monolayers

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20–40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.     

The phosphoinositide 3-kinase inhibitor LY294002, decreases aminoacyl-tRNA synthetases, chaperones and glycolytic enzymes in human HT-29 colorectal cancer cells

The proposed anticancer drug LY294002, inhibits phosphoinositide-3 kinase (PI3K) that initiates a signalling pathway often activated in colorectal cancer (CRC). The effects of LY294002 (10 μM, 48 h) on the cytosolic, mitochondrial and nuclear proteomes of human HT-29 CRC cells have been determined using iTRAQ (isobaric tag for relative and absolute quantitation) and tandem mass spectrometry (MS/MS). Analysis of cells treated with LY294002 identified 26 differentially abundant proteins that indicate several mechanisms of action. The majority of protein changes were directly or indirectly associated with Myc and TNF-α, previously implicated in CRC progression. LY294002 decreased the levels of 6 aminoacyl-tRNA synthetases (average 0.39-fold) required for protein translation, 5 glycolytic enzymes (average 0.37-fold) required for ATP synthesis, and 3 chaperones required for protein folding. There was a 3.2-fold increase in lysozyme C involved in protein-glycoside hydrolysis. LY294002 increased cytosolic p53 with a concomitant decrease in nuclear p53, suggesting transfer of p53 to the cytosol where apoptosis might be initiated via the intrinsic mitochondrial pathway. Protein changes described here suggest that the anti-angiogenic effects of LY294002 may be related to p53; the mutational status of p53 in CRC may be an important determinant of the efficacy of PI3K inhibitors for treatment.     

Abdominal paracentesis drainage attenuates intestinal mucosal barrier damage through macrophage polarization in severe acute pancreatitis

Abdominal paracentesis drainage (APD), as an effective treatment of severe acute pancreatitis (SAP) in clinical settings, can ameliorate intestinal barrier damage and the overall severity of SAP. However, the mechanism underlying therapeutic effects of APD on damaged intestinal mucosal barrier during SAP is still unclear. Here, SAP was induced by injecting 5% Na-taurocholate retrograde into the biliopancreatic duct of rats to confirm the benefits of APD on enteral injury of SAP and further explore the possible mechanism. Abdominal catheter was placed after SAP was induced in APD group. As control group, the sham group received no operation except abdominal opening and closure. By comparing changes among control group, sham group, and APD group, APD treatment obviously lowered the intestinal damage and reduced the permeation of intestinal mucosal barrier, which was evidenced by intestinal H&E staining, enteral expression of tight junction proteins, intestinal apoptosis measurement and detection of serum diamine oxidase, intestinal fatty acid binding protein and D-lactic acid. Furthermore, we found that APD polarized intestinal macrophages toward M2 phenotype by the determination of immunofluorescence and western blotting, and this accounts for the benefits of APD for intestinal injury in SAP. Importantly, the protective effect against intestinal injury by APD treatment was mediated through the inhibited ASK1/JNK pathway. In summary, APD improved the intestinal mucosal barrier damage in rats with SAP through an increasing portion of M2 phenotype macrophages in intestine via inhibiting ASK1/JNK pathway.     

Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes

We have investigated the effects of hyperosmolarity induced by sucrose on the fluid phase endocytosis of the fluorescent dye lucifer yellow CH (LY) and the endocytosis of 125I-asialo-orosomucoid (ASOR) by the galactosyl receptor system in isolated rat hepatocytes. Continuous uptake of LY by cells at 37 degrees C is biphasic, occurs for 3-4 h, and then plateaus. Permeabilized cells or crude membranes do not bind LY at 4 or 37 degrees C. Intact cells also do not accumulate LY at 4 degrees C. The rate and extent of LY accumulation are concentration- and energy-dependent, and internalized LY is released from permeabilized cells. Efflux of internalized LY from washed cells is also biphasic and occurs with halftimes of approximately 38 and 82 min. LY is taken up into vesicles throughout the cytoplasm and the perinuclear region with a distribution pattern typical of the endocytic pathway. LY, therefore, behaves as a fluid phase marker in hepatocytes. LY has no effect on the uptake of 125I-ASOR at 37 degrees C. The rate of LY uptake by cells in suspension is not affected for at least 30 min by up to 0.2 M sucrose. The rate of endocytosis of 125I-ASOR, however, is progressively inhibited by increasing the osmolality of the medium with sucrose (greater than 98% with 0.2 M sucrose; Oka and Weigel (1988) J. Cell. Biochem. 36, 169-183). Hyperosmolarity completely inhibits endocytosis of 125I-ASOR by the galactosyl receptor, whereas fluid phase endocytosis of LY is unaffected. Cultured hepatocytes contained about 100 coated pits/mm of apical membrane length as assessed by transmission electron microscopy. In the presence of 0.4 M sucrose, only 17 coated pits/mm of membrane were observed, an 83% decrease. Only a few percent of the total cellular fluid phase uptake in hepatocytes is due to the coated pit endocytic pathway. We conclude that the fluid phase and receptor-mediated endocytic processes must operate via two separate pathways.     

Measurement and chromatographic characterization of vasoactive intestinal peptide from guinea-pig enteric nerves

The material exhibiting immunoreactivity for vasoactive intestinal peptide in guinea-pig enteric nerves has been characterized by high-performance liquid chromatography in three modes: reversed-phase, cation-exchange and gel permeation. In each case a major portion of the material contained in acetic acid extracts of guinea-pig gut showed the same chromatographic properties as the synthetic porcine peptide of defined amino acid sequence. It is therefore concluded that this immunoreactive material is authentic vasoactive intestinal peptide. The study illustrates a number of the problems encountered in attempting to characterize, and measure reliably, peptides in tissue extracts.     

Inhibition of phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase leads to suppression of p34(cdc2) kinase activity and meiotic progression beyond the meiosis I stage in porcine oocytes surrounded with cumulus cells

In this study, the effects of U0126 that inhibits the activity of mitogen-activated protein (MAP) kinase kinase (MEK), and LY294002, which is a phosphatidylinositol (PI) 3-kinase inhibitor, on meiotic progression beyond the metaphase I (MI) stage in porcine oocytes were examined. Cumulus-oocyte complexes (COCs) were cultured for 22 h with 50 microM LY294002 or 10 microM U0126 following cultivation for the initial 22 h. MAP kinase activity in oocytes cultured with LY294002 or U0126 was significantly lower than that in control oocytes cultured for up to 44 h. U0126 and LY294002 significantly decreased p34(cdc2) kinase activity and the proportion of oocytes reaching the MII stage compared to those in control oocytes. Oocytes denuded after COCs had been cultured for 22 h were cultured further for 22 h with U0126 or LY294002. In the denuded oocytes, U0126 suppressed MAP kinase activity, p34(cdc2) kinase activity, and meiotic progression to the MII stage; however, LY294002 did not significantly affect the activity of these kinases and meiotic progression. These results suggest that increasing MAP kinase activity in oocytes via the PI 3-kinase signaling pathway in cumulus cells is involved in the stimulation of maturation promoting factor, leading to meiotic progression beyond the MI to MII stage in porcine oocytes.     

Distribution and characterisation of neuropeptide Y immunoreactivity in the brain and gastrointestinal tract of the rainbow trout,Oncorhynchus mykiss

1. Neuropeptide Y (NPY) immunoreactivity has been localised cytochemically in neuronal somata and fibres in rainbow trout brain, nerve fibres and mucosal epithelial endocrine cells within the gastrointestinal tract and in endocrine cells within pancreatic islets.2. Using a C-terminal specific NPY radioimmunoassay, immunoreactivity was detected in extracts of brain (519 pmol/g), cardiac stomach (37.9 pmol/g), pyloric stomach plus pancreas (37.9 ol/g) and intestine (29.2 pmol/g).3. Gel permeation and reverse-phase HPLC analysis of brain and intestinal extracts resolved a single NPY immunoreactive peptide.     

Tick-Borne Encephalitis Virus Replication,Intracellular Trafficking,and Pathogenicity in Human Intestinal Caco-2 Cell Monolayers

Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.     

Oviposition deterrents in larval frass of the cotton boll worm, Helicoverpa armigera (Lepidoptera: Noctuidae): chemical identification and electroantennography analysis

Oviposition deterrents in the frass of cotton bollworm (CBW), Helicoverpa armigera larvae fed on an artificial diet (FA) and on cotton Gossypium hirsutum leaves (FC) were investigated by behavioral bioassays and electroantennography analyses in the laboratory. It was found that a water suspension or a hexane extract of the frass FA or FC, in contrast to the corresponding foods, significantly deterred oviposition of conspecifics. When hexane extracts of the frass FA and FC were further partitioned into polar and neutral lipid fractions, two polar fractions significantly reduced oviposition. The neutral fraction from frass FC also exhibited significant deterrence, although the activity was much lower than that of the corresponding polar fraction. The polar lipid fractions contained several fatty acids, mainly palmitic and oleic acid at the ratio nearly 1:1. A blend of authentic fatty acids of the same composition found in frass FA or FC mimicked the deterring effect. Moreover, these fatty acids and their blend at the ratio found in frass FA or FC elicited significant electroantennogram responses and typical dose-response curves. Thus, it is suggested that CBW larvae may deploy two types of oviposition deterrents: a non-specific and a specific one. The former is a blend of fatty acids, independent of food and plays an important role in oviposition deterrence, whereas the latter may be produced only when the larvae feed on cotton leaves. The possible explanations of this deployment have also been discussed.     

The protein C pathway in inflammatory bowel disease: the missing link between inflammation and coagulation

Traditionally described as a major anti-coagulant system, the protein C (PC) pathway, consisting of thrombomodulin, the endothelial cell protein C receptor and activated PC (APC), is gaining increasing attention as an important regulator of microvascular inflammation. Although they possess several anti-inflammatory and cytoprotective functions, the expression and function of the components of the PC pathway is downregulated during inflammation. Recent evidence suggests that the PC pathway is defective in patients with inflammatory bowel disease (IBD) and that restoring its function has anti-inflammatory effects on cultured intestinal microvascular endothelial cells and in animal models of colitis. Here, we propose that the PC pathway has an important role in governing intestinal microvascular inflammation and might provide a novel therapeutic target in the management of IBD.     

Histamine H3 receptors modulate reactive hyperemia in rat gut.

Reactive hyperemia (RH) is an abrupt blood flow increase following release from mechanical occlusion of an artery, with restoration of intra-arterial pressure. The mechanism of this postocclusion increase in blood flow in the gut is multifactorial. Relaxation of intestinal resistance vessels, observed during RH, may involve myogenic, metabolic, hormonal and neurogenic factors. Evidence exists that histamine is an important endogenous mediator of various functions of the gut, including blood flow. The vascular effects of histamine in the intestinal circulation are due its agonistic action on histamine H1, H2 and H3 receptors. In the present study the hypothesis was tested that peripheral histamine H3 receptors are involved in the mediation of RH in the intestinal circulation. In anesthetized rats, anterior mesenteric artery blood flow (MBF) was determined with ultrasonic Doppler flowmeter, and arterial pressure (AP) was determined with a transducer. The increase in the volume of blood accumulating during RH (RH-volume), the peak increase of arterial blood flow (RH-peak response) and the duration of the hyperemia (RH-duration) were used to quantify RH after occluding the anterior mesenteric artery for 30, 60 and 120 s. Hyperemia parameters were determined before and after administration of the selective histamine H3 receptor antagonist clobenpropit. Pretreatment with clobenpropit was without any effect on control MBF and AP but significantly reduced most of RH responses. These findings support the hypothesis that histamine H3 receptors do not play any role in the control of intestinal vasculature at basal conditions but these receptors participate in the intestinal hyperemic reaction in response to complete temporal intestinal ischemia.     

Transport kinetics of leucine enkephalin across Caco-2 monolayers: quantitative analysis for contribution of enzymatic and transport barrier

In this study, we determined the activities of four aminopeptidases such as aminopeptidase B (APB), M (APM), N (APN) and dipeptidylpeptidase IV (DPP IV) in Caco-2 cells and compared with those in the rat intestinal mucosae. The activities of APB, APM and APN appeared to be highest in rat small intestinal mucosa, while DPP IV activity was much higher in Caco-2 cells than that in the rat intestinal mucosa. Next the inhibitory effects of various protease inhibitors were examined in Caco-2 homogenate. Three tested inhibitors, bacitracin, amastatin and puromycin, effectively inhibited the activities of APM, APN and DPP IV except for APB. Further, we quantitatively evaluated the permeation and degradation properties of leucine enkephalin (Leu-Enk) in the presence or absence of inhibitors in Caco-2 monolayer system. Leu-Enk had a high degradation clearance (CLd) and a low permeation clearance (CLp) in Caco-2 monolayers. This finding indicates that the very rapid degradation of Leu-Enk on the apical side of Caco-2 monolayers was due to aminopeptidases. However, these protease inhibitors besides sodium glycocholate were able to reduce the CLd values markedly, thereby increasing the permeation amount of Leu-Enk across Caco-2 monolayers. In particular, amastatin significantly decreased the CLd value and increased the CLp value. This enhanced CLp value was further increased by the coadministration with an absorption enhancer, EDTA or laurylmaltoside. These findings are relevant to the oral administration of peptide drugs and to developing an efficient oral delivery system.     

Autophagy is decreased in mesenteric fat tissue but not in intestinal mucosae of patients with Crohn’s disease

Crohn??s disease (CD) is a chronic intestinal disease with a multifactorial etiology. Recently, a role for mesenteric fat has been proposed in CD pathophysiology, since fat hypertrophy is detected close to the affected intestinal area; however, there are few studies regarding autophagy and the hypertrophied mesenteric tissue in CD. To evaluate autophagy-related proteins in intestinal mucosae and mesenteric fat of patients with CD and controls, patients with ileocecal CD (CD Group) and with non-inflammatory disease (FC Group) selected for surgery were studied. Expression of LC3-II was determined by immunoblotting of protein extracts. In addition, beclin-1, LC3 and Atg16-L1 RNA levels were measured using RT-PCR. The expression of LC3-II was significantly lower in the mesenteric tissue and higher in intestinal mucosae of CD when compared to controls. However, mRNA expression of autophagy-related proteins was similar when comparing the mesenteric fat groups. These findings suggest a defect in autophagy activation in the mesenteric fat tissue of CD individuals, which could be involved in the maintenance of the inflammatory process.     

Akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase-3 after polyamine depletion

Apoptosis plays a critical role in the maintenance of gut mucosal homeostasis and is regulated by numerous factors including polyamines. Although the exact roles of polyamines in apoptotic pathway are still unclear, inhibition of polyamine synthesis promotes the resistance of intestinal epithelial cells to apoptosis. Akt is a serine-threonine kinase that has been established as an important intracellular signaling in regulating cell survival. The current studies test the hypothesis that polyamines are involved in the control of Akt activity in normal intestinal epithelial cells (IEC-6 line) and that activated Akt mediates suppression of apoptosis following polyamine depletion. Depletion of cellular polyamines by alpha-difluoromethylornithine induced levels of phosphorylated Akt and increased Akt kinase activity, although it had no effect on expression of total Akt, pERK, p38, and Bcl-2 proteins. This activated Akt was associated with both decreased levels of active caspase-3 and increased resistance to tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Inactivation of Akt by either treatment with LY294002 or ectopic expression of a dominant negative Akt mutant (DNMAkt) not only enhanced the caspase-3 activation in polyamine-deficient cells but also prevented the increased resistance to tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Phosphorylation of glycogen synthase kinase-3, a downstream target of Akt, was also increased in alpha-difluoromethylornithine-treated cells, which was prevented by inactivation of Akt by LY294002 or DNMAkt overexpression. These results indicate that polyamine depletion induces the Akt activation mediating suppression of apoptosis via inhibition of caspase-3 in normal intestinal epithelial cells.