A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.     

Resveratrol Enhances Palmitate-Induced ER Stress and Apoptosis in Cancer Cells

Background

Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several in vitro and in vivo studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines.

Principal Findings

We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity.

Conclusions

Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by ER stress through a CHOP-mediated apoptotic process and may represent a potential anticancer strategy.     

Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells

Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. However, the signaling pathways and mechanisms linking lipid accumulation to cell death are poorly understood. The present study examined the hypothesis that saturated fatty acids disrupt endoplasmic reticulum (ER) homeostasis and promote apoptosis in liver cells via accumulation of ceramide. H4IIE liver cells were exposed to varying concentrations of saturated (palmitate or stearate) or unsaturated (oleate or linoleate) fatty acids. ER homeostasis was monitored using markers of the ER stress response pathway, including phosphorylation of IRE1alpha and eIF2alpha, splicing of XBP1 mRNA, and expression of molecular chaperone (e.g., GRP78) and proapoptotic (CCAAT/enhancer-binding protein homologous protein) genes. Apoptosis was monitored using caspase activity and DNA laddering. Palmitate and stearate induced ER stress, caspase activity, and DNA laddering. Inhibition of caspase activation prevented DNA laddering. Unsaturated fatty acids did not induce ER stress or apoptosis. Saturated fatty acids increased ceramide concentration; however, inhibition of de novo ceramide synthesis did not prevent saturated fatty acid-induced ER stress and apoptosis. Unsaturated fatty acids rescued palmitate-induced ER stress and apoptosis. These data demonstrate that saturated fatty acids disrupt ER homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation.     

Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells

Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.     

Enhanced synthesis of saturated phospholipids is associated with ER stress and lipotoxicity in palmitate treated hepatic cells

High levels of saturated FAs (SFAs) are acutely toxic to a variety of cell types, including hepatocytes, and have been associated with diseases such as type 2 diabetes and nonalcoholic fatty liver disease. SFA accumulation has been previously shown to degrade endoplasmic reticulum (ER) function leading to other manifestations of the lipoapoptotic cascade. We hypothesized that dysfunctional phospholipid (PL) metabolism is an initiating factor in this ER stress response. Treatment of either primary hepatocytes or H4IIEC3 cells with the SFA palmitate resulted in dramatic dilation of the ER membrane, coinciding with other markers of organelle dysfunction. This was accompanied by increased de novo glycerolipid synthesis, significant elevation of dipalmitoyl phosphatidic acid, diacylglycerol, and total PL content in H4IIEC3 cells. Supplementation with oleate (OA) reversed these markers of palmitate (PA)-induced lipotoxicity. OA/PA cotreatment modulated the distribution of PA between lipid classes, increasing the flux toward triacylglycerols while reducing its incorporation into PLs. Similar trends were demonstrated in both primary hepatocytes and the H4IIEC3 hepatoma cell line. Overall, these findings suggest that modifying the FA composition of structural PLs can protect hepatocytes from PA-induced ER stress and associated lipotoxicity.     

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.     

Palmitate exacerbates bisphenol A toxicity via induction of ER stress and mitochondrial dysfunction

Combined exposure to dietary nutrients and environmental chemicals may elicit significantly different physiological effects than single exposures. Exposure to dietary saturated fats and environmental toxins is a physiologically-significant dual exposure that is particularly associated with lower socioeconomic status, potentially placing these individuals at heightened risk of xenobiotic toxicities. However, no prior studies have examined interactions between specific lipids and environmental xenobiotics in modulating cellular health. Using primary mouse embryonic fibroblasts, we have discovered that prior exposure to the saturated fatty acid, palmitate, exacerbates cellular toxicity associated with the industrial plasticizer, bisphenol A (BPA). Cell death upon BPA exposure following palmitate pre-treatment was greater than that occurring with either exposure alone. Mechanistically, cell death was preceded by increased endoplasmic reticulum stress and loss of mitochondrial membrane potential in palmitate plus BPA exposed cells, leading to increased caspase-3 cleavage and subsequent apoptosis. Interestingly, inclusion of the unsaturated fatty acid, oleate, along with palmitate during the pre-treatment period completely abrogated the ER stress, mitochondrial toxicity, and cell death induced by subsequent exposure to BPA. Thus, our data identify for the first time an important interaction between a fatty acid and an environmental toxin and have implications for developing nutritional interventions to mitigate the deleterious effects of such xenobiotic exposures.     

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78

Alterations in Ca(2+) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.     

Loss of cardiolipin in palmitate-treated GL15 glioblastoma cells favors cytochrome c release from mitochondria leading to apoptosis

Unlike oleate and linoleate, palmitate induced mitochondrial apoptosis in GL15 glioblastoma cells. Decrease in membrane potential in a subpopulation of mitochondria of palmitate-treated cells was revealed using the 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide probe. The diminished ability to reduce a tetrazolium salt indicated an impairment of mitochondrial function. Up to 50% cytochrome c (cyt c ) was detached from the inner mitochondrial membrane and released outside mitochondria in palmitate-treated cells, whereas no release was detected after oleate and linoleate treatments. Cyt c release into the cytosol was followed by caspase 3 activation. Released cyt c and caspase 3 activity were not affected by neutral and acid sphingomyelinase inhibitors and by the inhibitor of serine palmitoyltransferase cycloserine, indicating that apoptosis was independent of the ceramide pathway, nor the mitochondrial pro-apoptotic AIF or Bcl-2/Bax factors appeared to be involved in the effect. Utilization of palmitate by GL15 cells altered phospholipid composition. Cardiolipin (CL), the lipid involved in cyt c interaction with the inner mitochondrial membrane, was decreased and highly saturated. This produced an imbalance in hydrophilic/hydrophobic interactions underlying the anchorage of cyt c , by weakening the hydrophobic component and facilitating detachment of the protein and activation of downstream processes. The primary role of CL was explored by supplying GL15 with exogenous CL through a fusion process of CL liposomes with cell plasma membrane. Fused CL moved to mitochondria, as detected by nonylacridine orange probe. Enrichment of mitochondrial membranes with CL prior to palmitate treatment of cells caused decreased cyt c release and caspase 3 activity.     

ER signaling in unfolded protein response

Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death. To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER). This is known as the unfolded protein response (UPR). ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system. Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system. Hrd3p interacts with and stabilizes Hrd1p. We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p. Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER. HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death. These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.     

Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress

In response to endoplasmic reticulum (ER) stress, cells launch homeostatic and protective responses, but can also activate cell death cascades. A 54 kDa integral ER membrane protein called Herp was identified as a stress-responsive protein in non-neuronal cells. We report that Herp is present in neurons in the developing and adult brain, and that it is regulated in neurons by ER stress; sublethal levels of ER stress increase Herp levels, whereas higher doses decrease Herp levels and induce apoptosis. The decrease in Herp protein levels following a lethal ER stress occurs prior to mitochondrial dysfunction and cell death, and is mediated by caspases which generate a 30-kDa proteolytic Herp fragment. Mutagenesis of the caspase cleavage site in Herp enhances its neuroprotective function during ER stress. While suppression of Herp induction by RNA interference sensitizes neural cells to apoptosis induced by ER stress, overexpression of Herp promotes survival by a mechanism involving stabilization of ER Ca(2+) levels, preservation of mitochondrial function and suppression of caspase 3 activation. ER stress-induced activation of JNK/c-Jun and caspase 12 are reduced by Herp, whereas induction of major ER chaperones is unaffected. Herp prevents ER Ca(2+) overload under conditions of ER stress and agonist-induced ER Ca(2+) release is attenuated by Herp suggesting a role for Herp in regulating neuronal Ca(2+) signaling. By stabilizing ER Ca(2+) homeostasis and mitochondrial functions, Herp serves a neuroprotective function under conditions of ER stress.     

Experimental and computational studies investigating trehalose protection of HepG2 cells from palmitate-induced toxicity

Understanding the mechanism of saturated fatty acid-induced hepatocyte toxicity may provide insight into cures for diseases such as obesity-associated cirrhosis. Trehalose, a nonreducing disaccharide shown to protect proteins and cellular membranes from inactivation or denaturation caused by different stress conditions, also protects hepatocytes from palmitate-induced toxicity. Our results suggest that trehalose serves as a free radical scavenger and alleviates damage from hydrogen peroxide secreted by the compromised cells. We also observe that trehalose protects HepG2 cells by interacting with the plasma membrane to counteract the changes in membrane fluidity induced by palmitate. The experimental results are supported by molecular dynamics simulations of model cell membranes that closely reflect the experimental conditions. Simulations were performed to understand the specific interactions between lipid bilayers, palmitate, and trehalose. The simulations results reveal the early stages of how palmitate induces biophysical changes to the cellular membrane and the role of trehalose in protecting the membrane structure.     

Alteration of Endoplasmic Reticulum Lipid Rafts Contributes to Lipotoxicity in Pancreatic β-Cells

Chronic saturated fatty acid exposure causes β-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger β-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 β-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating β-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.     

Mitochondrial degeneration and not apoptosis is the primary cause of embryonic lethality in ceramide transfer protein mutant mice

Ceramide transfer protein (CERT) functions in the transfer of ceramide from the endoplasmic reticulum (ER) to the Golgi. In this study, we show that CERT is an essential gene for mouse development and embryonic survival and, quite strikingly, is critical for mitochondrial integrity. CERT mutant embryos accumulate ceramide in the ER but also mislocalize ceramide to the mitochondria, compromising their function. Cells in mutant embryos show abnormal dilation of the ER and degenerating mitochondria. These subcellular changes manifest as heart defects and cause severely compromised cardiac function and embryonic death around embryonic day 11.5. In spite of ceramide accumulation, CERT mutant mice do not die as a result of enhanced apoptosis. Instead, cell proliferation is impaired, and expression levels of cell cycle–associated proteins are altered. Individual cells survive, perhaps because cell survival mechanisms are activated. Thus, global compromise of ER and mitochondrial integrity caused by ceramide accumulation in CERT mutant mice primarily affects organogenesis rather than causing cell death via apoptotic pathways.     

AMPK activation prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 signaling and endoplasmic reticulum stress response

Lipid accumulation is a central event in the development of chronic metabolic diseases, including obesity and type 2 diabetes, but the mechanisms responsible for lipid accumulation are incompletely understood. This study was designed to investigate the mechanisms for excess nutrient-induced lipid accumulation and whether activation of AMP-activated protein kinase (AMPK) prevents the hepatic lipid accumulation in excess nutrient-treated HepG2 cells and high fat diet (HFD)-fed mice. Exposure of HepG2 cells to high levels of glucose or palmitate induced the endoplasmic reticulum (ER) stress response, activated sterol regulatory element-binding protein-1 (SREBP-1), and enhanced lipid accumulation, all of which were sensitive to ER stress inhibitor and gene silencing of eukaryotic initiation factor 2α. The increases in ER stress response and lipid accumulation were associated with activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Inhibition of mTORC1 signaling attenuated the ER stress response and lipid accumulation induced by high glucose or by deletion of tuberous sclerosis 2. In addition, AMPK activation prevented the mTORC1 activation, ER stress response, and lipid accumulation. This effect was mimicked or abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Finally, treatment of HFD-fed mice with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside inhibited the mTORC1 pathway, suppressed the ER stress response, and prevented insulin resistance and hepatic lipid accumulation. We conclude that activation of AMPK prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 and ER stress response.     

The intracellular voyage of cholera toxin: going retro

Cholera toxin (CT) and related AB₅-subunit toxins move from the plasma membrane through the trans-Golgi and endoplasmic reticulum (ER) to the cytosol of host cells. The toxins exploit a specific glycolipid pathway rather than a protein pathway. They bind glycolipids that associate with lipid rafts at the cell surface, which carry the toxins retrograde to the Golgi and ER. In the ER, the A1-chain of the CT unfolds and enters the cytosol by hijacking the cellular machinery that enables misfolded proteins to cross the membrane for degradation by the proteasome, a process termed retro-translocation. Upon entering the cytosol, the A1-chain rapidly refolds, avoids the proteasome and induces toxicity.     

Endoplasmic Reticulum Enrollment in Alzheimer’s Disease

Alzheimer??s disease (AD) poses a huge challenge for society and health care worldwide as molecular pathogenesis of the disease is poorly understood and curative treatment does not exist. The mechanisms leading to accelerated neuronal cell death in AD are still largely unknown, but accumulation of misfolded disease-specific proteins has been identified as potentially involved. In the present review, we describe the essential role of endoplasmic reticulum (ER) in AD. Despite the function that mitochondria may play as the central major player in the apoptotic process, accumulating evidence highlights ER as a critical organelle in AD. Stress that impairs ER physiology leads to accumulation of unfolded or misfolded proteins, such as amyloid ?? (A??) peptide, the major component of amyloid plaques. In an attempt to ameliorate the accumulation of unfolded proteins, ER stress triggers a protective cellular mechanism, which includes the unfolded protein response (UPR). However, when activation of the UPR is severe or prolonged enough, the final cellular outcome is pathologic apoptotic cell death. Distinct pathways can be activated in this process, involving stress sensors such as the JNK pathway or ER chaperones such as Bip/GRP94, stress modulators such as Bcl-2 family proteins, or even stress effectors such as caspase-12. Here, we detail the involvement of the ER and associated stress pathways in AD and discuss potential therapeutic strategies targeting ER stress.     

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.