Iron homeostasis is maintained in the brain,but not the liver,following mild hypoxia

Abstract

Alterations in iron metabolism or oxidative damage in response to hypoxic incidents have been examined following re-oxygenation of the hypoxic tissue. To understand the consequences of decreased tissue oxygen on iron load, metal-catalyzed redox activity and oxidative modifications in isolation from re-oxygenation, the present study exposed mice to either normoxia, or mild hypoxia (380 Torr; ～10% normobaric oxygen) where the tissue was not allowed to re-oxygenate prior to examination. Brain, liver and skeletal muscle were examined for Fe³⁺ load, metal-catalyzed redox activity and oxidative modifications to proteins (N^?-(carboxymethyl)lysine), lipids (4-hydroxynonenal pyrrole) and nucleic acids (8-hydroxyguanosine). Hypoxia induced a 43% increase in the iron content of the liver (P < 0.001) as determined by ICP-MS and a 3.8-fold increase in Fe³⁺ load (P < 0.001) as determined by Perl's stain. There was a corresponding 2-fold increase in metal-catalyzed redox activity (P < 0.01) in the liver, but no change in the expression of oxidative markers. In contrast, non-significant increases in Fe³⁺ and metal-catalyzed redox activity were observed in the cerebral cortex, and molecular and granular layers of the hippocampus and cerebellum. Interestingly, hypoxia significantly decreased oxidative modifications to proteins and lipids, but not nucleic acids in most brain regions examined. In addition, hypoxia did not alter the Fe content of skeletal muscle, or the contents of Zn, Cu, Ni or Mn in liver, skeletal muscle, cerebral cortex or hippocampus. Together, these results indicate that there is a tighter regulation of iron metabolism in the brain than the liver, which limits the redistribution of Fe³⁺ following hypoxia.     

Iron homeostasis is dysregulated,but the iron-hepcidin axis is functional,in chronic liver disease

BackgroundPerturbations in iron homeostasis have been reported to be associated with irreversible liver injury in chronic liver disease (CLD). However, it is not clear whether liver dysfunction per se underlies such dysregulation or whether other factors also contribute to it. This study attempted to examine the issues involved.MethodsPatients diagnosed to have chronic liver disease (n = 63), who underwent a medically-indicated upper gastrointestinal endoscopy, were the subjects of this study. Patients with dyspepsia, who underwent such a procedure, and were found to have no endoscopic abnormalities, were used as control subjects (n = 49). Duodenal mucosal samples were obtained to study mRNA and protein levels of duodenal proteins involved in iron absorption. A blood sample was also obtained for estimation of hematological, iron-related, inflammatory and liver function-related parameters.ResultsPatients with CLD had impaired liver function, anemia of inflammation and lower serum levels of hepcidin than control subjects. Gene (mRNA) expression levels of duodenal ferroportin and duodenal cytochrome b (proteins involved in iron absorption) were decreased, while that of divalent metal transporter–1 (DMT-1) was unchanged. Protein expression of DMT-1 was, however, decreased while that of ferroportin was unchanged. In the CLD group, serum hepcidin was predicted independently by serum ferritin and hemoglobin, but not by C-reactive protein (a marker of inflammation). CLD patients with serum ferritin greater than 300 μg/dL had significantly greater liver dysfunction (as indicated by significantly higher serum concentrations of bilirubin, AST and ALT, and MELD scores), higher serum concentrations of CRP and hepcidin, and higher ferroportin protein expression, than those with serum ferritin ≤ 300 μg/dL.ConclusionsIn patients with CLD, anemia of inflammation and low serum hepcidin levels were found to paradoxically co-exist. Expression of duodenal proteins involved in iron absorption were either decreased or unaltered in these patients. The hepcidin response to higher body iron levels and/or inflammation appeared to be functional in these patients, despite the presence of liver disease.     

Extraocular muscle in merosin-deficient muscular dystrophy: cation homeostasis is maintained but is not mechanistic in muscle sparing

Extraocular muscle is uniquely spared from damage in merosin-deficient congenital muscular dystrophy. Using a murine model, we have tested the hypothesis that the maintenance of calcium homeostasis is mechanistic in extraocular muscle protection. Atomic absorption spectroscopy has demonstrated a strong correlation between the perturbation of calcium homeostasis in hindlimb muscle that is severely damaged and the absence of changes in calcium in extraocular muscle. If, as in other skeletal muscles, extraocular muscle fibers are destabilized by merosin deficiency, we would expect an increase in total muscle calcium coupled with an adaptive response in the high capacity/speed of the sarcoplasmic reticulum of the eye muscle. However, we have not observed the expected increases in total muscle calcium content, Ca²⁺-ATPase activity, Na⁺/Ca²⁺ exchanger content, or smooth ER Ca²⁺-ATPase content that are predicted by this model. Instead, these results indicate that the increased membrane permeability that characterizes, and is potentially mechanistic in, myofiber degeneration in muscular dystrophy does not occur in merosin-deficient extraocular muscle. Thus, the high-capacity calcium-scavenging systems are not primarily responsible for extraocular muscle protection in muscular dystrophy.     

Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury

Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl₄. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1⁺ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1⁺ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl₄-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of “activated” sinusoidal HSC, but it is a marker of “activated” (myo)fibroblasts found in portal areas and in scar tissue. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The HAL1 function on Na+ homeostasis is maintained over time in salt-treated transgenic tomato plants, but the high reduction of Na+ in leaf is not associated with salt tolerance

To achieve a deeper knowledge on the function of HAL1 gene in tomato ( Solanum lycopersicum ) plants submitted to salt stress, in this study, we studied the growth and physiological responses to high salt stress of T3 transgenic plants (an azygous line without transgene and both homozygous and hemizygous lines for HAL1 ) proceeding from a primary transformant with a very high expression level of HAL1 gene. The homozygous plants for HAL1 gene did not increase their salt tolerance in spite of an earlier and higher reduction of the Na⁺ accumulation in leaves, being moreover the Na⁺ homeostasis maintained throughout the growth cycle. The greater ability of the homozygous line to regulate the Na⁺ transport to the shoot to long term was even shown in low accumulation of Na⁺ in fruits. By comparing the homozygous and hemizygous lines, a higher salt tolerance in the hemizygous line, with respect to the homozygous line, was observed on the basis of fruit yield. The Na⁺ homeostasis and osmotic homeostasis were also different in homozygous and hemizygous lines. Indeed, the Na⁺ accumulation rate in leaves was greater in hemizygous than in homozygous line after 35 days of 100 m M NaCl treatment and only at the end of growth cycle did the hemizygous line show leaf Na⁺ levels similar to those found in the homozygous line. With respect to the osmotic homeostasis, the main difference between lines was the different contribution of inorganic and organic solutes to the leaf osmotic balance. Taken together, these results suggest that the greater Na⁺ exclusion ability of the homozygous line overexpressing HAL1 induces a greater use of organic solutes for osmotic balance, which seems to have an energy cost and hence a growth penalty that reverts negatively on fruit yield.     

Extracellular potassium homeostasis in the cat medulla during progressive brain hypoxia

Brain extracellular potassium [( K+]ec) in the ventral respiratory group of the medulla and the phrenic neurogram were recorded in anesthetized vagotomized peripherally chemodenervated ventilated cats during progressive isocapnic carbon monoxide (CO) hypoxia. During hypoxia, the phrenic neurogram was progressively depressed and became silent when arterial O2 content (CaO2) was reduced by 62 +/- 3% (SE). Gasping was seen in the phrenic neurogram when CaO2 was reduced by 78 +/- 1%. Medullary [K+]ec, an indicator of energy production failure due to O2 insufficiency, was 3.2 +/- 0.4 mM before hypoxia and was statistically unchanged at the onset of phrenic apnea during CO hypoxia (4 +/- 0.7 mM). By the onset of gasping, [K+]ec had increased to 6.1 +/- 1 mM, a value that tended to be different from control (P less than 0.1). After initiation of gasping, the rate of rise of [K+]ec increased, and [K+]ec reached a maximum value of 14.3 +/- 2.7 mM before hypoxia was terminated. With reoxygenation, [K+]ec returned to control levels within 20 min. On the basis of these results, we have drawn two major conclusions. 1) Hypoxic depression to the point of phrenic apnea does not appear to be caused by medullary energy insufficiency as measured by loss of [K+]ec homeostasis. 2) The rapid rise in [K+]ec in the medulla that characterizes severe hypoxia is closely associated with the onset of gasping in the phrenic neurogram, suggesting that gasping may serve as a marker for loss of medullary ionic homeostasis and thus onset of medullary energy insufficiency during hypoxia.     

Novel isoenzyme of 2-oxoglutarate dehydrogenase is identified in brain, but not in heart

2-Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multienzyme OGDH complex (OGDHC) whose malfunction is associated with neurodegeneration. The essential role of this complex in the degradation of glucose and glutamate, which have specific significance in brain, raises questions about the existence of brain-specific OGDHC isoenzyme(s). We purified OGDHC from extracts of brain or heart mitochondria using the same procedure of poly(ethylene glycol) fractionation, followed by size-exclusion chromatography. Chromatographic behavior and the insufficiency of mitochondrial disruption to solubilize OGDHC revealed functionally significant binding of the complex to membrane. Components of OGDHC from brain and heart were identified using nano-high performance liquid chromatography electrospray tandem mass spectrometry after trypsinolysis of the electrophoretically separated proteins. In contrast to the heart complex, where only the known OGDH was determined, the band corresponding to the brain OGDH component was found to also include the novel 2-oxoglutarate dehydrogenase-like (OGDHL) protein. The ratio of identified peptides characteristic of OGDH and OGDHL was preserved during purification and indicated comparable quantities of the two proteins in brain. Brain OGDHC also differed from the heart complex in the abundance of the components, lower apparent molecular mass and decreased stability upon size-exclusion chromatography. The functional competence of the novel brain isoenzyme and different regulation of OGDH and OGDHL by 2-oxoglutarate are inferred from the biphasic dependence of the overall reaction rate versus 2-oxoglutarate concentration. OGDHL may thus participate in brain-specific control of 2-oxoglutarate distribution between energy production and synthesis of the neurotransmitter glutamate.     

delta-, but not mu-, opioid receptor stabilizes K(+) homeostasis by reducing Ca(2+) influx in the cortex during acute hypoxia

Past work has shown that delta-opioid receptor (DOR) activation by [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) attenuated the disruption of K(+) homeostasis induced by hypoxia or oxygen-glucose deprivation (OGD) in the cortex, while naltrindole, a DOR antagonist blocked this effect, suggesting that DOR activity stabilizes K(+) homeostasis in the cortex during hypoxic/ischemic stress. However, several important issues remain unclear regarding this new observation, especially the difference between DOR and other opioid receptors in the stabilization of K(+) homeostasis and the underlying mechanism. In this study, we asked whether DOR is different from micro-opioid receptors (MOR) in stabilizing K(+) homeostasis and which membrane channel(s) is critically involved in the DOR effect. The main findings are that (1) similar to DADLE (10 microM), H-Dmt-Tic-NH-CH (CH(2)--COOH)-Bid (1-10 microM), a more specific and potent DOR agonist significantly attenuated anoxic K(+) derangement in cortical slice; (2) [D-Ala(2), N-Me-Phe(4), glycinol(5)]-enkephalin (DAGO; 10 microM), a MOR agonist, did not produce any appreciable change in anoxic disruption of K(+) homeostasis; (3) absence of Ca(2+) greatly attenuated anoxic K(+) derangement; (4) inhibition of Ca(2+)-activated K(+) (BK) channels with paxilline (10 microM) reduced anoxic K(+) derangement; (5) DADLE (10 microM) could not further reduce anoxic K(+) derangement in the Ca(2+)-free perfused slices or in the presence of paxilline; and (6) glybenclamide (20 microM), a K(ATP) channel blocker, decreased anoxia-induced K(+) derangement, but DADLE (10 microM) could further attenuate anoxic K(+) derangement in the glybenclamide-perfused slices. These data suggest that DOR, but not MOR, activation is protective against anoxic K(+) derangement in the cortex, at least partially via an inhibition of hypoxia-induced increase in Ca(2+) entry-BK channel activity.     

Genomic imprinting in Drosophila is maintained by the products of Suppressor of variegation and trithorax group,but not Polycomb group,genes

Genomic imprinting is a form of epigenetic inheritance that is characterized by differential expression of a gene depending on its parental origin. The mini-X chromosome Dp(1;f)LJ9 in Drosophila shows this type of classical imprinting; when transmitted by the maternal parent genes on this chromosome are fully expressed, but when the chromosome is transmitted by the male parent at least three genes are subject to silencing, resulting in a variegated expression pattern. Chemical and environmental modifiers of position-effect variegation have been shown to alter the somatic maintenance of the imprint. To extend these observations, several mutations in chromatin-associated proteins were examined for their effect on imprinting on the Dp(1;f)LJ9 mini-X chromosome. Effects on establishment and maintenance were independently assessed by genetically associating the mutations in chromatin modifiers with the mini-X chromosome in either the parents, where the imprint is established, or the progeny, in which the imprint must be maintained. Nine Suppressor of variegation [ Su(var)] mutations, including alleles of the Su(var)2-5 gene, which encodes the well characterized heterochromatin-associated protein HP1, abolished maintenance but not the establishment of the imprint. Mutant alleles of two genes in the trithorax group ( trx-G), brahma and trithorax, showed a maternal-effect enhancement of the paternal imprint. Surprisingly, however, with the exception of an Enhancer of Polycomb [ E(Pc)] allele, none of the Polycomb-group ( Pc-G) mutations tested affected the imprint. Thus, the maintenance of this imprint relies on the wild-type products of Su(var) and trx-G, but not Pc-G, genes. Finally, none of the mutations tested affected the maintenance of the maternal imprint or the establishment of either the maternal or paternal imprint, suggesting that the maternal and paternal imprints depend on different molecular processes and that imprint establishment and maintenance are independently regulated.     

Iron overload in Hepc1(-/-) mice is not impairing glucose homeostasis

Diabetes Mellitus is found with increasing frequency in iron overload patients with hemochromatosis. In these conditions, the pancreas shows predominant iron overload in acini but also islet beta-cells. We assess glucose homeostasis status in iron-overloaded hepcidin-deficient mice. These mice presented with heavy pancreatic iron deposits but only in the acini. The beta-cell function was found unaffected with a normal production and secretion of insulin. The mutant mice were not diabetic, responded as the control group to glucose and insulin challenges, with no alteration of insulin signalling in the muscle and the liver. These results indicate that, beta-cells iron deposits-induced decreased insulin secretory capacity might be of primary importance to trigger diabetes in hemochromatosic patients.     

Rat liver carbonic anhydrase I is not sexually dimorphic but is estrogen repressible

Carbonic anhydrase (CA) isozymes CAII and CAIII are known to exhibit sexual dimorphism in rat liver, and the levels o f these isozymes are affected by sex hormones. In this paper we show that the isozyme CAI is present at low levels in rat liver, with no difference in concentration between male and female rats. Estrogen and diethylstilbestrol reduce CAI levels in both sexes.     

The cost of evolved constitutive lac gene expression is usually,but not always,maintained during evolution of generalist populations

Beneficial mutations can become costly following an environmental change. Compensatory mutations can relieve these costs, while not affecting the selected function, so that the benefits are retained if the environment shifts back to be similar to the one in which the beneficial mutation was originally selected. Compensatory mutations have been extensively studied in the context of antibiotic resistance, responses to specific genetic perturbations, and in the determination of interacting gene network components. Few studies have focused on the role of compensatory mutations during more general adaptation, especially as the result of selection in fluctuating environments where adaptations to different environment components may often involve trade‐offs. We examine whether costs of a mutation in lacI, which deregulated the expression of the lac operon in evolving populations of Escherichia coli bacteria, were compensated. This mutation occurred in multiple replicate populations selected in environments that fluctuated between growth on lactose, where the mutation was beneficial, and on glucose, where it was deleterious. We found that compensation for the cost of the lacI mutation was rare, but, when it did occur, it did not negatively affect the selected benefit. Compensation was not more likely to occur in a particular evolution environment. Compensation has the potential to remove pleiotropic costs of adaptation, but its rarity indicates that the circumstances to bring about the phenomenon may be peculiar to each individual or impeded by other selected mutations.     

Retinol-binding protein mRNA is induced by estrogen in the kidney but not in the liver

Vitamin A is mobilized from the liver and transported in plasma as retinol bound to retinol-binding protein (RBP). In addition to the liver, several extrahepatic tissues including the kidney have been shown to contain RBP mRNA. A study was conducted to explore the role of sex hormones in the regulation of RBP mRNA levels in the kidney compared to those in the liver. Treatment of female rats with a single dose of testosterone or chronic treatment with testosterone had only a slight effect on the steady-state level of RBP mRNA in the kidney and the liver. However, treatment of male rats with estrogen caused an increase in the steady-state level of RBP mRNA in the kidney but not in the liver. A single injection of 17 beta-estradiol, either 1.0 or 0.1 micrograms/g body weight, resulted in a rapid rise in the level of RBP mRNA in the kidney which was maximal at 3-6 h (fivefold induction) after treatment. In addition, treatment of ovariectomized female rats with estrogen also resulted in a rapid rise in the accumulated level of RBP mRNA in the kidney while having no influence in the liver. Finally, studies using the anti-estrogen drug, hydroxytamoxifen, resulted in blockage of the estrogen-related induction of RBP mRNA in the kidney, suggesting that the induction of RBP mRNA in the kidney by estrogen may be mediated by the nuclear estrogen receptor. Taken together these data suggest that the regulation of RBP mRNA, levels in the liver and kidney, at least with respect to estrogen, is different.     

Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.

Episodic hypoxia evokes a sustained augmentation of respiratory motor output known as long-term facilitation (LTF). Phrenic LTF is prevented by pretreatment with the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin. We tested the hypothesis that 5-HT receptor activation is necessary for the induction but not maintenance of phrenic LTF. Peak integrated phrenic nerve activity (integralPhr) was monitored for 1 h after three 5-min episodes of isocapnic hypoxia (arterial PO(2) = 40 +/- 2 Torr; 5-min hyperoxic intervals) in four groups of anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats [1) control (n = 11), 2) ketanserin pretreatment (2 mg/kg iv; n = 7), and ketanserin treatment 0 and 45 min after episodic hypoxia (n = 7 each)]. Ketanserin transiently decreased integralPhr, but it returned to baseline levels within 10 min. One hour after episodic hypoxia, integralPhr was significantly elevated from baseline in control and in the 0- and 45-min posthypoxia ketanserin groups. Conversely, ketanserin pretreatment abolished phrenic LTF. We conclude that 5-HT receptor activation is necessary to initiate (during hypoxia) but not maintain (following hypoxia) phrenic LTF.     

ATP-NGF-complex, but not NGF, is the neuroprotective ligand

We have shown previously that nerve growth factor (NGF) requires only low nanomolar ATP concentrations in the cell culture medium to protect cortical rat neurons (CRN) from cellular damage induced by staurosporine (STS). We have also demonstrated before that NGF and other growth factors form stable non-covalent complexes with ATP. Here we demonstrated that 8N₁ATP–NGF, but not NGF, protected CRN against damage. The photo-reactive ATP derivative 8N₃ATP was incubated with NGF and was trapped in its position by UV irradiation forming a covalent bond. The cross-link with a molar ratio of 1:1 (8N₁ATP:NGF) was confirmed by mass spectrometry. Circular dichroism experiments revealed that 8N₁ATP altered the secondary structure of NGF in the same way as ATP did. Covalently bound 8N₁ATP–NGF was shown to be stable in the presence of the ATP-hydrolyzing enzyme alkaline phosphatase while the non-covalent ATP–NGF-complex dissociated with the removal of free ATP from the solution. 8N₁ATP–NGF protected CRN against damage by STS independently of free ATP in the culture medium. These results suggest that the ATP–NGF-complex, but not NGF, is the active ligand.     

Allometric scaling of fatty acyl chains in fowl liver,lung and kidney,but not in brain phospholipids

The phospholipid (PL) fatty acyl chain (FA) composition (mol%) was determined in the kidney, liver, lung and brain of 8 avian species ranging in body mass from 150 g (Japanese quail, Coturnix coturnix japonica) to 19 kg (turkey, Meleagris gallopavo). In all organs except the brain, docosahexaenoic acid (C22:6 n3, DHA) was found to show a negative allometric scaling (allometric exponent: B = ? 0.18; ? 0.20 and ? 0.24, for kidney, liver and lung, respectively). With minor inter-organ differences, smaller birds had more n3 FAs and longer FA chains in the renal, hepatic and pulmonary PLs. Comparing our results with literature data on avian skeletal muscle, liver mitochondria and kidney microsomes and divergent mammalian tissues, the present findings in the kidney, liver and lung PLs seem to be a part of a general relationship termed “membranes as metabolic pacemakers”. Marked negative allometric scaling was found furthermore for the tissue malondialdehyde concentrations in all organs except the brain (B = ? 0.17; ? 0.13 and ? 0.05, respectively). In the liver and kidney a strong correlation was found between the tissue MDA and DHA levels, expressing the role of DHA in shaping the allometric properties of membrane lipids.     

Drinking,but not feeding,is opiate-sensitive in hamsters

The long-lasting opiate antagonist, naltrexone (NTX), was examined for its effects on various types of consummatory behavior in male golden hamsters and rats. Rat, but not hamster, 24 hr food and water intakes were significantly decreased by four daily NTX (10.0 mg/kg) injections. Hamsters displayed a minimal night to day feeding ratio compared to rats. hamsters increased food intake following insulin (50 U/kg) administration, but not after 24 hr food deprivation (FD) or 2-deoxy-D-glucose (2-DG; 800 mg/kg) injections. NTX (1.0 and 10 mg/kg) had no effect on feeding, but markedly attenuated hamster drinking induced by 48 hr water deprivation or hypertonic saline injection. Dexamethasone (DEX), a glucocorticoid which depletes pituitary β-endorphin and produces anorexia in rats, had no effect on daily hamster intake. Since the normal feeding profile of the hamster is similar to that of naloxone and DEX-treated rats, hamsters appear to lack an opiate-sensitive feeding system. In contrast, stimulated drinking behavior of hamsters operates through an opiate-sensitive mechanism. Thus, there are marked species differences concerning the involvement of endogenous opioids is consummatory behavior.     

Iron homeostasis in the lung

Iron is essential for many aspects of cellular function. However, it also can generate oxygen-based free radicals that result in injury to biological molecules. For this reason, iron acquisition and distribution are tightly regulated. Constant exposure to the atmosphere results in significant exposure of the lungs to catalytically active iron. The lungs have a mechanism for detoxification to prevent associated generation of oxidative stress. Those same proteins that participate in iron uptake in the gut are also employed in the lung, to transport iron intracellularly and sequester it in an inactive form within ferritin. The release of metal is expedited (as transferrin and ferritin) from lung tissue to the respiratory lining fluid for clearance by the mucocilliary pathway or to the reticuloendothelial system for long-term storage. This pathway is likely to be the major method for the control of oxidative stress presented to the respiratory tract.     

Compensatory hepatic regeneration after mild, but not fulminant, intraperitoneal sepsis in rats

Sepsis is the leading cause of death in surgical intensive care units. Although both mild sepsis secondary to cecal ligation and single puncture (CLP) and fulminant, double puncture CLP (2CLP) may provoke hepatocyte death, we hypothesize that regeneration compensates for cell death after CLP but not 2CLP. In male Sprague-Dawley rats, hepatic necrosis, as determined by serum alpha-glutathione S-transferase (alpha-GST) levels, was significantly but equally elevated over time after both CLP and 2CLP. Apoptosis, evaluated using both terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and morphological examination, was minimal after both CLP and 2CLP. Regeneration, assayed by staining tissue for incorporation of exogenously administered bromodeoxyuridine, was present after CLP but not after 2CLP. To further substantiate impaired regeneration, steady-state levels of mRNAs encoding JunB, LRF-1, and cyclin D1 were determined. After 2CLP, the absence of JunB, LRF-1, and cyclin D1 mRNAs confirmed failed activation of the mitogen-activated protein kinase-linked proliferative pathway and progression through the cell cycle. Therefore, failed hepatocyte regeneration may be a manifestation of hepatic dysfunction in fulminant sepsis.