Increased brain endothelial nitric oxide synthase expression in thiamine deficiency: relationship to selective vulnerability

Thiamine deficiency results in selective neuronal cell death in thalamic structures. Previous studies provide evidence for a role implicating nitric oxide (NO) in the pathogenesis of cell death due to thiamine deficiency. In order to ascertain the origin of increased NO in the thiamine deficient brain, expression of endothelial nitric oxide synthase isoform (eNOS), was measured in the medial thalamus and in the inferior colliculus and compared to the frontal cortex (a spared region) of rats in which thiamine deficiency was induced through a feeding protocol of thiamine-deficient diet concomitant with daily administration of pyrithiamine, a central thiamine antagonist. eNOS mRNA and protein expression were significantly increased as a function of the severity of neurological impairment and the degree of neuronal cell loss in the medial thalamus and in the inferior colliculus. These findings suggest that the vascular endothelium is a major site of NO production in the brain in thiamine deficiency and that eNOS-derived NO could account for the selective damage to the thalamic structures that are observed in this particular disorder.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine-deficiency encephalopathy:

Chronic thiamine deprivation in the rat leads to selective neuropathological damage in brainstem structures whereas treatment with the central thiamine antagonist, pyrithiamine, results in more widespread damage. In order to further elucidate the neurochemical mechanisms responsible for this selective damage, the thiamine-dependent enzyme complex pyruvate dehydrogenase (PDHC) was measured in 10 brain structures in the rat during progression of thiamine deficiency produced by chronic deprivation or by pyrithiamine treatment. Feeding of a thiamine-deficient diet to adult rats resulted in 5–7 weeks in ataxia and loss of righting reflex accompanied by decreased blood transketolase activities. PDHC activities were selectively decreased by 15–30% in midbrain and pons (lateral vestibular nucleus). Thiamine treatment of symptomatic rats led to reversal of neurological signs and to concomitant reductions of the cerebral PDHC abnormalities. Daily pyrithiamine treatment led within 3 weeks to loss of righting reflex and convulsions and to decreased blood transketolase of a comparable magnitude to that observed in chronic thiamine-deprived rats. No significant regional alterations of PDHC, however, were observed in pyrithiamine-treated rats.     

Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions

Culture of neuroblastoma cells in the presence of low thiamine concentration (6 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 µM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional. (Mol Cell Biochem 174: 121–124, 1997)     

JNK1 is inactivated during thiamine deficiency-induced apoptosis in human neuroblastoma cells

Thiamine deficiency results in selective neuronal damage. A number of mechanisms have been proposed to account for brain damage associated with thiamine deficiency and to account for the focal nature of the loss of neurons. One proposed mechanism is programmed cell death. We found efficient induction of apoptosis in human neuroblastoma cells when the cells were deprived of thiamine. Although extensive mitochondrial damage was seen, the release of cytochrome c was not the triggering mechanism for thiamine deficiency-induced apoptosis. Instead, the activity of the cJun amino terminal kinase Jnk1 was lost, and this loss correlated temporally with induction of apoptosis. The loss was specific for Jnk1; Jnk2/3 activity remained unchanged. Loss of Jnk1 activity was not found in lymphoblasts, a cell type that did not undergo apoptosis when deprived of thiamine. These findings suggest that thiamine deficiency results in a cellular stress that brings about the loss of Jnk1 activity and the loss of its function of protecting cells from programmed cell death. We postulate that focal sensitivity to thiamine deficiency results, in part, from specific neuronal cell types being susceptible to the inactivation of Jnk1 in response to depletion of cellular thiamine.     

Alterations in Neurotransmitter Receptor Binding in Discrete Areas of the Copper-Deficient Rat Brain

Abstract: Neonatal copper deficiency produced alterations in central neurotransmitter receptors that were selective with respect both to brain region and to neurotransmitter receptor type. Both high- and low-affinity dopamine receptor densities in the corpus striatum were significantly lowered, 55% and 29%, respectively, when expressed on a wet weight basis. There was a significant decrease in the level of muscarinic receptors in the striatum whether expressed on the basis of wet weight (50%) or protein (27%). A smaller reduction in muscarinic receptor density was observed in the cortex, whereas there was no effect of copper deficiency in the cerebellum. The treatment did not change β-adrenergic receptor binding in either the cortex or cerebellum. The affinities of the receptors for the ligands was not affected by the low-copper diet. It was previously reported that copper deficiency produces regionally specific decreases in the concentrations of dopamine and norepinephrine. The greatest reduction occurred in the concentration of dopamine in the corpus striatum. The results from both studies suggest that copper deficiency in post-weanling rats may induce a selective morphological lesion.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine-deficiency encephalopathy 2. α-ketoglutarate dehydrogenase

Chronic thiamine deprivation in the rat leads to selective neuropathological damage to pontine structures. Onset of neurological symptoms of thiamine deprivation (ataxia, loss of righting reflex) was accompanied by selective decreases (of the order of 30%) in the activity of -ketoglutarate dehydrogenase (KGDH) in lateral vestibular nucleus and hypothalamus. Enzyme activities were decreased to a lesser extent in medulla oblongata, striatum and hippocampus and were unchanged in other brain structures. No changes in KGDH occurred prior to the onset of neurological signs of thiamine deprivation. Administration of the central thiamine antagonist, pyrithiamine, results within 3 weeks in loss of righting reflex and convulsions and in more widespread neuropathological changes than those observed following thiamine deprivation. KGDH activities were found to be substantially diminished in all brain regions studied following pyrithiamine treatment with most severe changes occurring in brain regions found to be vulnerable to pyrithiamine (lateral vestibular nucleus, hypothalamus, midbrain, medullapons). In some cases, KGDH changes preceded the appearance of neurological symptoms of pyrithiamine treatment. Such decreases in KGDH may explain previous findings of region-selective changes in energy metabolism and of decreased synthesis of glucose-derived neurotransmitters (acetylcholine, GABA, glutamate) in pyrithiamine-treated rat brain. Thiamine administration to symptomatic pyrithiamine treated rats resulted in reversal of neurological signs of encephalopathy and in normalisation of defective KGDH activity in all brain regions. These findings suggest that the reversible neurological symptoms associated with Wernicke's Encephalopathy in man likely result from region-selective impairment of KGDH.     

Loss of [3H]kainate and of NMDA-displaceable [3H]glutamate binding sites in brain in thiamine deficiency: Results of a quantitative autoradiographic study

Previous studies suggest that alterations of brain glutamate synthesis and release occur in experimental thiamine deficiency. In order to assess the integrity of post-synaptic glutamatergic receptors in thiamine deficiency, binding sites for [³H]glutamate (displaced by NMDA), [³H]-kainate, and [³H]quisqualate (AMPA sites) were evaluated using Quantitative Receptor Autoradiography in rat brain following 14 days of treatment with the central thiamine antagonist pyrithiamine. Compared to pair-fed controls, brains of symptomatic thiamine-deficient animals contained significantly fewer NMDA-displaceable binding sites in cerebral cortex, medial septum and hippocampus. It has been suggested that NMDA-receptor mediated glutamate excitotoxicity plays a role in the pathogenesis of neuronal loss in thiamine deficiency. If such is the case, the selective loss of NMDA binding sites in cerebral cortex and hippocampus offers a possible explanation for the relative nonvulnerability of these brain regions to pyrithiamine-induced thiamine deficiency. [³H]quisqualate (AMPA) binding sites were unchanged in all brain regions of pyrithiamine-treated rats whereas [³H]kainate sites were significantly reduced in density in medial and lateral thalamus. The decline in these binding sites may be due to neuronal loss in pyrithiamine-induced thiamine deficiency. Alterations of glutamatergic synaptic function involving both NMDA and kainate receptor subclasses could contribute to the pathogenesis of neurological dysfunction in Wernicke's Encephalopathy in humans.     

Abolition of reperfusion-induced arrhythmias in hearts from thiamine-deficient rats

Extensive work has been done regarding the impact of thiamine deprivation on the nervous system. In cardiac tissue, chronic thiamine deficiency is described to cause changes in the myocardium that can be associated with arrhythmias. However, compared with the brain, very little is known about the effects of thiamine deficiency on the heart. Thus this study was undertaken to explore whether thiamine deprivation has a role in cardiac arrhythmogenesis. We examined hearts isolated from thiamine-deprived and control rats. We measured heart rate, diastolic and systolic tension, and contraction and relaxation rates. Whole cell voltage clamp was performed in rat isolated cardiac myocytes to measure L-type Ca(2+) current. In addition, we investigated the global intracellular calcium transients by using confocal microscopy in the line-scan mode. The hearts from thiamine-deficient rats did not degenerate into ventricular fibrillation during 30 min of reperfusion after 15 min of coronary occlusion. The antiarrhythmogenic effects were characterized by the arrhythmia severity index. Our results suggest that hearts from thiamine-deficient rats did not experience irreversible arrhythmias. There was no change in L-type Ca(2+) current density. Inactivation kinetics of this current in Ca(2+)-buffered cells was retarded in thiamine-deficient cardiac myocytes. The global Ca(2+) release was significantly reduced in thiamine-deficient cardiac myocytes. The amplitude of caffeine-releasable Ca(2+) was lower in thiamine-deficient myocytes. In summary, we have found that thiamine deprivation attenuates the incidence and severity of postischemic arrhythmias, possibly through a mechanism involving a decrease in global Ca(2+) release.     

Thiamine Deficiency-Induced Partial Necrosis and Mitochondrial Uncoupling in Neuroblastoma Cells Are Rapidly Reversed by Addition of Thiamine

Abstract: Culture of neuroblastoma cells in a medium of low-thiamine concentration (6 n M ) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate-dependent α-ketoglutarate dehydrogenase activity is impaired. When thiamine (10 µ M ) is added to the cells, the O₂ consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate-independent succinate dehydrogenase, is also able to restore a normal O₂ consumption (with respiratory control) in digitonin-permeabilized thiamine-deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernicke's encephalopathy.     

Role of mitochondrial dysfunction and oxidative stress in the pathogenesis of selective neuronal loss in Wernicke’s encephalopathy

Thiamine deficiency results in Wernicke’s encephalopathy and is commonly encountered in chronic alcoholism, gastrointestinal diseases, and HIV AIDS. The earliest metabolic consequence of thiamine deficiency is a selective loss in activity of the thiamine diphosphate-dependent enzyme α-ketoglutarate dehydrogenase (α-KGDH), a rate-limiting tricarboxylic acid cycle enzyme. Thiamine deficiency is characterized neuropathologically by selective neuronal cell death in the thalamus, pons, and cerebellum. The cause of this region-selective neuronal loss is unknown, but mechanisms involving cellular energy failure, focal lactic acidosis, and NMDA receptor-mediated excitotoxicity have classically been implicated. More recently, evidence supports a role for oxidative stress. Evidence includes increased endothelial nitric oxide synthase, nitrotyrosine deposition, microglial activation, and lipid peroxidation. Reactive oxygen species production results in decreased expression of astrocytic glutamate transporters and decreased activities of α-KGDH, resulting in an amplification of cell death mechanisms in thiamine deficiency.     

Increased "peripheral-type" benzodiazepine receptor sites and mRNA in thalamus of thiamine-deficient rats.

"Peripheral-type" benzodiazepine receptors (PTBRs) are highly expressed on the outer mitochondrial membrane of several types of glial cells. In order to further elucidate the nature of the early glial cell changes in thiamine deficiency, PTBR sites and PTBR mRNA were measured in thalamus, a brain structure which is particularly vulnerable to thiamine deficiency, of thiamine-deficient rats at presymptomatic and symptomatic stages of deficiency. PTBR sites were measured using an in vitro binding technique and the selective radio ligand [3H]-PK11195. PTBR gene expression was measured by RT-PCR using oligonucleotide primers based upon the published sequence of the cloned rat PTBR. Microglial and astrocytic changes in thalamus due to thiamine deficiency were assessed using immunohistochemistry and antibodies to specific microglial (ED-1) and astrocytic (GFAP) proteins respectively. Significant increases of [3H]-PK11195 binding sites and concomitantly increased PTBR mRNA were observed in thalamus at the symptomatic stage of thiamine deficiency, coincident with severe neuronal cell loss and increased GFAP-immunolabelling (indicative of reactive gliosis). Positron Emission Tomography using 11C-PK11195 could provide a novel approach to the diagnosis and assessment of the extent of thalamic damage due to thiamine deficiency in humans with Wernicke's Encephalopathy.     

eNOS gene deletion restores blood-brain barrier integrity and attenuates neurodegeneration in the thiamine-deficient mouse brain

Wernicke's encephalopathy is a cerebral disorder caused by thiamine (vitamin B₁) deficiency (TD). Neuropathologic consequences of TD include region-selective neuronal cell loss and blood-brain barrier (BBB) breakdown. Early increased expression of the endothelial isoform of nitric oxide synthase (eNOS) occurs selectively in vulnerable brain regions in TD. We hypothesize that region-selective eNOS induction in TD leads to altered expression of tight junction proteins and BBB breakdown. In order to address this issue, TD was induced in C57BL/6 wild-type (WT) and eNOS^−/− mice by feeding a thiamine-deficient diet and treatment with the thiamine antagonist pyrithiamine. Pair-fed control mice were fed the same diet with additional thiamine. In medial thalamus of TD-WT mice (vulnerable area), increased heme oxygenase-1 and S -nitrosocysteine immunostaining was observed in vessel walls, compared to pair-fed control-WT mice. Concomitant increases in IgG extravasation, decreases in expression of the tight junction proteins occludin, zona occludens-1 and zona occludens-2, and up-regulation of matrix metalloproteinase-9 in endothelial cells were observed in the medial thalamus of TD-WT mice. eNOS gene deletion restored these BBB alterations, suggesting that eNOS-derived nitric oxide is a major factor leading to cerebrovascular alterations in TD. However, eNOS gene deletion only partially attenuated TD-related neuronal cell loss, suggesting the presence of mechanisms additional to BBB disruption in the pathogenesis of these changes.     

A quantitative autoradiographic study of muscarinic cholinergic receptor subtypes in the brains of pyrithiamine-treated rats

Previous studies describe decreased acetylcholine synthesis in brain as well as neurobehavioural evidence for a central muscarinic cholinergic deficit in pyrithiamine-induced thiamine-deficient rats. In order to further evaluate this possibility, quantitative autoradiographic procedures using [³H]quinuclidinyl benzilate (for total muscarinic binding sites), [³H]pirenzepine (for muscarinic M₁ sites) and [³H]AF-DX 384 (for muscarinic M₂ sites) were performed at early (presymptomatic) and late (symptomatic) stages of thiamine deficiency induced in rats by administration of the central thiamine antagonist, pyrithiamine. No significant alterations in densities of M₁, M₂ or total muscarinic binding sites were observed in any brain structure evaluated at either early or late stages of thiamine deficiency. These findings do not support a major role for modifications of muscarinic cholinergic function in the pathogenesis of the neurological symptoms of thiamine deficiency.     

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.     

Presynaptic terminals in hyaline cells of normal and overstimulated chick inner ears

Hyaline cells are non-sensory epithelial cells of the vibrating part of the basilar membrane of chicks; they receive an extensive efferent innervation. Although these anatomical features suggest roles in auditory transduction, very little is known about the function of these cells. One possible way to understand function is by lesion experiments. We used synapsin-specific antibodies to study changes that occur in the pattern of efferent innervation in hyaline cells after lesion of the sensory epithelium induced by acoustic overstimulation. We found only small changes in hyaline cells after such trauma. These included a small increase in size and a small decrease in density of nerve terminals on hyaline cells. This suggests that hyaline cells and their nerve terminals are less susceptible to acoustic trauma than hair cells. Using neurofilament-specific antibodies we found little or no trauma-induced change in the density of nerve fibres that cross the basilar papilla and reach the hyaline cell region. This finding suggested that trauma to the hair cells does not necessarily lead to changes in the efferent fibres that cross the papilla and extend into the hyaline cell region. Using the trauma and the morphological parameters studied here, it appears that a moderate lesion in the hair cell region in the avian inner ear does not influence the hyaline cells or their innervation.     

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.     

Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats

AimsThiamine is an important cofactor present in many biochemical reactions, and its deprivation can lead to heart dysfunction. Little is known about the influence of thiamine deprivation on the electrophysiological behavior of the isolated heart cells and information about thiamine deficiency in heart morphology is controversial. Thus, we decided to investigate the major repolarizing conductances and their influence in the action potential (AP) waveform as well as the changes in the heart structure in a set of thiamine deficiency in rats.Main methodsUsing the patch-clamp technique, we investigated inward (I_K1) and outward K⁺ currents (I_to), T-type and L-type Ca²⁺ currents and APs. To evaluate heart morphology we used hematoxylin and eosin in transversal heart sections.Key findingsThiamine deficiency caused a marked decrease in left ventricle thickness, cardiomyocyte number, cell length and width, and membrane capacitance. When evaluating I_to we did not find difference in current amplitude; however an acceleration of I_to inactivation was observed. I_K1 showed a reduction in the amplitude and slope conductance, which implicated a less negative resting membrane potential in cardiac myocytes isolated from thiamine-deficient rats. We did not find any difference in L-type Ca²⁺ current density. T-type Ca²⁺ current was not observed. In addition, we did not observe significant changes in AP repolarization.SignificanceBased on our study we can conclude that thiamine deficiency causes heart hypotrophy and not heart hypertrophy. Moreover, we provided evidence that there is no major electrical remodeling during thiamine deficiency, a feature of heart failure models.     

Blood thiamine and thiamine phosphate ester concentrations in alcoholic and non-alcoholic liver diseases.

Thiamine state was investigated in patients with alcoholic liver disease, patients with various non-alcoholic liver diseases, and controls using a direct technique (thiochrome assay) to measure thiamine, thiamine monophospate, and the active coenzyme thiamine pyrophosphate in whole blood after isolating the fractions by ion exchange chromatography. Overall nutrition was similar in all groups as assessed by anthropometry, and no patient had clinical evidence of thiamine deficiency. There was no significant difference among the groups in mean concentration of any form of thiamine. The scatter was much greater in patients with alcoholic liver disease but only 8.7% had biochemical thiamine deficiency (defined as a blood concentration of the active coenzyme greater than 2 SD below the mean control value). An unexpected finding was of abnormally high total thiamine concentrations (greater than 2 SD above the mean control value) in 17.4% of patients with alcoholic liver disease, the highest concentrations being found in two patients with severe alcoholic hepatitis and cirrhosis. The ratio of phosphorylated to unphosphorylated thiamine was calculated as an index of phosphorylation and, although the mean did not differ significantly among the groups, the range was greatest in alcoholic liver disease. The lowest ratios occurred in the two patients with severe alcoholic hepatitis, but neither had evidence of thiamine pyrophosphate deficiency. Contrary to studies using indirect assay techniques, these results suggest that thiamine deficiency is unusual in well nourished patients with alcoholic liver disease. The new finding of unexpectedly high thiamine concentrations in some patients may be due to abnormalities of hepatic storage or release in liver disease, particularly in severe alcoholic hepatitis. There was no convincing evidence of impaired thiamine phosphorylation in any patients with liver disease. Conclusions from studies using indirect assays on the prevalence and mechanisms of thiamine deficiency in liver diseases may not be valid.     

Transganglionic regulation of the primary sensory neuron

Central terminals of the primary sensory neurons depend on the integrity of the retrograde transport mechanism within the peripheral axon. Whenever retrograde transport is impaired (either by injury or by blockade induced by perineural application of microtubule inhibitors) central terminals undergo transganglionic degenerative atrophy (TDA), characterized by depletion of substance P, somatostatin, FRAP (fluoride resistant acid phosphatase), TMPase (thiamine monophosphatase) and lectin-binding fucose-terminated glyco-conjugates. The TDA is essentially a failure of the central terminals to bind the above genuine marker substances. TDA-inflicted central terminals undergo a slowly proceeding ultrastructural deterioration, accompanied by derangement of the dorsal root potential, reflecting decreased functional activity of synaptic transmission between first and second-order cells. One of the important trophic substances carried by retrograde axoplasmic transport to dorsal root ganglion cells is nerve growth factor (NGF); blockade of NGF transport results in TDA; conversely, locally applied NGF delays or prevents TDA.     

Arginine deficiency in preconfluent intestinal Caco-2 cells modulates expression of proteins involved in proliferation, apoptosis, and heat shock response

Arginine is classified as a conditionally essential amino acid required exogenously during catabolic disease states and periods of rapid growth, both characterized by increased arginine utilization. Arginine plays an important role in the intestine, where it is extensively metabolized, and enhances its immune-supportive function and mucosal repair. Cell proliferation is important for the latter process. This study aimed for a better molecular insight in the response to arginine deprivation/supplementation of preconfluent and 5-day-confluent, differentiated Caco-2 intestinal cells. The potential of citrulline to counteract the effects of arginine deprivation was investigated in preconfluent cells. 2-DE combined with MALDI-TOF-MS and the antibody microarray technology were applied. Evidence is provided that arginine deficiency modulates the protein expression profiles of preconfluent Caco-2 cells differently than that of postconfluent differentiated cells. In preconfluent cells, certain proteins changed in direct response to arginine deficiency, whereas other proteins did not, but instead responded during the recovery phase after an arginine/citrulline resupplementation. The protein changes suggest that arginine deprivation decreases cell proliferation and heat shock protein expression, and enhances the cells susceptibility to apoptosis. These processes are critical for proper cell function, and hence a state of arginine deficiency can be detrimental for intestinal cells which proliferate actively in vivo.