Effect of pulsatile growth hormone administration to the growth-restricted fetal sheep on somatotrophic axis gene expression in fetal and placental tissues.

We have previously reported (Bauer MK, Breier BH, Bloomfield FH, Jensen EC, Gluckman PD, and Harding JE. J Endocrinol 177: 83-92, 2003) that a chronic pulsatile infusion of growth hormone (GH) to intrauterine growth-restricted (IUGR) ovine fetuses increased fetal circulating IGF-I levels without increasing fetal growth. We hypothesized a cortisol-induced upregulation of fetal hepatic GH receptor (GH-R) mRNA levels, secondary increases in IGF-I mRNA levels, and circulating IGF-I levels, but a downregulation of the type I IGF receptor (IGF-IR) as an explanation. We, therefore, measured mRNA levels of genes of the somatotrophic axis by real-time RT-PCR in fetal and placental tissues of fetuses with IUGR (induced by uteroplacental embolization from 110- to 116-days gestation) that received either a pulsatile infusion of GH (total dose 3.5 mg/day) or vehicle from 117-126 days and in control fetuses (n = 5 per group). Tissues were collected at 127 days (term, 145 days). Fetal cortisol concentrations were significantly increased in IUGR fetuses. However, in liver, GH-R, but not IGF-I or IGF-IR, mRNA levels were decreased in both IUGR groups. In contrast, in placenta, GH-R, IGF-I, and IGF-IR expression were increased in IUGR vehicle-infused fetuses. GH infusion further increased placental GH-R and IGF-IR, but abolished the increase in IGF-I mRNA levels. GH infusion reduced IGF-I expression in muscle and increased GH-R but decreased IGF-IR expression in kidney. IUGR increased hepatic IGF-binding protein (IGFBP)-1 and placental IGFBP-2 and -3 mRNA levels with no further effect of GH infusion. In conclusion, the modest increases in circulating cortisol concentrations in IUGR fetuses did not increase hepatic GH-R mRNA expression and, therefore, do not explain the increased circulating IGF-I levels that we found with GH infusion, which are likely due to reduced clearance rather than increased production. We demonstrate tissue-specific regulation of the somatotrophic axis in IUGR fetuses and a discontinuity between GH-R and IGF-I gene expression in GH-infused fetuses that is not explained by alterations in phosphorylated STAT5b.

Amniotic IGF-I supplementation of growth-restricted fetal sheep alters IGF-I and IGF receptor type 1 mRNA and protein levels in placental and fetal tissues.

We have previously reported that chronic intra-amniotic supplementation of the late gestation growth-restricted (IUGR) ovine fetus with IGF-I (20 microg/day) increased gut growth but reduced liver weight and circulating IGF-I concentrations. Here we report mRNA and protein levels of IGF-I, the type 1 IGF receptor (IGF-1R) and IGF-binding proteins (IGFBP)-1, -2 and -3 in fetal gut, liver, muscle and placenta from fetuses in that earlier study in an attempt to explain these contrasting results. mRNA and protein were extracted from tissues obtained at post mortem at 131 days of gestation (term, 145 days) from three groups of fetuses (control, IUGR+saline and IUGR+IGF-I, n=9 per group). Control fetuses were unembolised and untreated. In the IUGR groups, growth restriction was induced from 113 to 120 days by placental embolisation; from 120 to 130 days fetuses were treated with daily intra-amniotic injections of either saline or 20 microg IGF-I. mRNA was measured by RT-PCR or real-time RT-PCR, and protein by Western blot. In liver, muscle and placenta, IGF-I mRNA and protein levels were reduced by between 8 and 30% in IGF-I-treated fetuses compared with saline-treated fetuses and controls with no change in IGF-1R mRNA or protein levels. In contrast, in the gut, IGF-I mRNA and protein levels were not significantly altered with IGF-I treatment, but IGF-1R levels were increased, especially in the jejunum. Immunolocalisation demonstrated that IGF-1R expression was confined to the luminal aspect of the gut. mRNA levels of all three IGFBPs were reduced in the gut of IGF-I-treated fetuses, but hepatic expression was significantly increased. These data demonstrated tissue-specific regulation of IGF-I, IGF-1R and IGFBPs-1, -2 and -3 in response to intra-amniotic IGF-I supplementation, though the underlying mechanisms remain obscure.

Chronic pulsatile infusion of growth hormone to growth-restricted fetal sheep increases circulating fetal insulin-like growth factor-I levels but not fetal growth.

Intra-uterine growth restriction (IUGR) is a major cause of perinatal mortality and morbidity. Postnatally, growth hormone (GH) increases growth, increases circulating insulin-like growth factor (IGF)-I levels, and alters metabolism. Our aim was to determine if GH infusion to IUGR fetal sheep would alter fetal growth and metabolism, and thus provide a potential intra-uterine treatment for the IUGR fetus. We studied three groups of fetuses: control, IUGR+ vehicle and IUGR+GH (n=5 all groups). IUGR was induced by repeated embolisation of the placental vascular bed between 110 and 116 days of gestation (term=145 days). GH (3.5 mg/kg/day) or vehicle was infused in a pulsatile manner from 117 to 127 days of gestation. Embolisation reduced fetal growth rate by 25% (P<0.01) and reduced the weight of the fetal liver (20%), kidney (23%) and thymus (31%; all P<0.05). GH treatment further reduced the weight of the fetal kidneys (32%) and small intestine (35%; both P<0.04), but restored the relative weight of the fetal thymus and liver (P<0.05). Embolisation decreased fetal plasma IGF-I concentrations (48%, P<0.001) and increased IGF binding protein 1 (IGFBP-1) concentrations (737%, P<0.002). GH treatment restored fetal plasma IGF-I concentrations to control levels, while levels in IUGR+vehicle fetuses stayed low (P<0.05 vs control). IGFBP-1 and IGFBP-2 concentrations were about sevenfold lower in amniotic fluid than in fetal plasma, but amniotic and plasma concentrations were closely correlated (r=0.75, P<0.0001 and r=0.55 P<0.0001 respectively). Embolisation transiently decreased fetal blood oxygen content (40%, P<0.002), and increased blood lactate concentrations (213%, P<0.04). Both returned to pre-embolisation levels after embolisation stopped, but blood glucose concentrations declined steadily in IUGR+vehicle fetuses. GH treatment maintained fetal blood glucose concentrations at control levels. Our study shows that GH infusion to the IUGR fetal sheep restores fetal IGF-I levels but does not improve fetal growth, and further reduces the fetal kidney and intestine weights. Thus, fetal GH therapy does not seem a promising treatment stratagem for the IUGR fetus.

Daily low-dose administration of growth hormone secretagogue stimulates pulsatile growth hormone secretion and elevates plasma insulin-like growth factor-1 levels in pigs.

Repeated administration of growth hormone secretagogues (GHSs) has proven to be a delicate matter owing to development of tolerance. The aim of the present study was to define conditions during which the responsiveness to the orally active NN703 was maintained over several days. Growing pigs were fitted with stomach and vascular catheters, permitting unstressed intragastric administrations and blood sampling. NN703 or vehicle was administered once daily. When NN703 was given at a dose of 18 mg/kg, there was a massive acute increase in plasma growth hormone (GH) levels, but this was only seen on the first day of administration. A dose of 1.8 mg/kg did not cause a significant acute increase in plasma GH concentrations, whereas stimulation of pulsatile GH release was sustained over a 4-d period. During the first 7 h following injection of vehicle, the area under the curve of plasma GH was 1211+/-144 (microg/[L x 7 h]), but increased to 1770+/-269 and 1824+/-198 (microg/[L x 7 h]) on the first and fourth day of NN703 administration, respectively. Deconvolution analysis of the 7-h profiles revealed that the GH mass per burst as well as the GH burst amplitude were significantly (p < 0.001) increased during treatment with NN703, which led to an increase in pulsatile GH secretion rate (p < 0.001). Insulin-like growth factor-1 plasma concentrations increased steadily during NN703 administration (p < 0.01) and decreased after termination of treatment. The sustained increase in GH pulsatility observed with low-dose NN703 treatment suggests that development of tolerance to this GHS may be obviated by minimization of dose.

Exogenous GH infusion to late-gestational fetal sheep does not alter fetal growth and metabolism.

The role of GH in the regulation of fetal growth and metabolism in late gestation is not well defined. The aim of this study was to determine the effects of exogenous GH infusion on fetal growth and feto-placental metabolism in the normally growing late-gestation fetal sheep. Eleven fetuses received pulsatile GH infusion (3.5 mg/day) for 10 days while 12 control fetuses received vehicle. The GH infusion was given as a continuous infusion (2.5 mg/day) plus an additional pulsatile component (30 pulses equivalent to 1 mg/day) designed to mimic the natural pattern of GH secretion. Fetal GH infusion raised the circulating fetal concentrations of GH threefold, but did not change fetal concentrations of IGF-I, IGF-binding protein-3, insulin or ovine placental lactogen. GH-treated fetuses had blood urea concentrations 15% lower than controls (P<0.05) and glucose uptake 18% lower per kg fetal weig! ht (P=0.06). There were no other differences attributable to fetal GH infusion in feto-placental metabolism, placental function or placental blood flow. GH-treated fetuses were larger than controls at postmortem (weight+13%, P<0.01; girth+5%, P<0.01; crown-rump length+3%, P<0.05). However, there were no differences between groups in measures of fetal growth (increment in chest girth and hindlimb length). GH-treated fetuses had heavier mothers and when maternal weight was included as a covariate in the analysis, there was no significant difference between treatment groups that could be attributed to GH treatment. GH infusion to normal fetal sheep does not appear to have a significant effect on feto-placental metabolism or fetal growth.

The cardiac Fas (APO-1/CD95) Receptor/Fas ligand system : relation to diastolic wall stress in volume-overload hypertrophy in vivo and activation of the transcription factor AP-1 in cardiac myocytes.

BACKGROUND: Fas (APO-1/CD95) is a transmembrane receptor belonging to the tumor necrosis factor receptor superfamily. Cross-linking of Fas by Fas ligand (FasL), a tumor necrosis factor-alpha-related cytokine, promotes apoptosis and/or transcription factor activation in a highly cell-type-specific manner. The biological consequences of Fas activation in cardiomyocytes and the regulation of Fas and FasL abundance in the myocardium in vivo remain largely unknown. METHODS AND RESULTS: As shown by immunohistochemistry, Fas was expressed on the sarcolemma of cardiomyocytes in left ventricular tissue sections. Moreover, FasL was constitutively expressed in the myocardium and in isolated cardiomyocytes, as revealed by reverse transcription polymerase chain reaction and Western blotting. Left ventricular abundance of Fas but not FasL was upregulated in a rat model of compensated volume-overload hypertrophy and was closely related to diastolic but not systolic wall stress as determined by MRI. Cardiomyocyte apoptosis was not enhanced in volume-overload hypertrophy despite the increased expression of Fas and the presence of FasL in the myocardium. Moreover, injection of mice with an agonistic anti-Fas antibody promoted hepatocyte but not cardiomyocyte apoptosis in vivo. Stimulation of isolated cardiomyocytes with recombinant FasL promoted an activation of the transcription factor AP-1 as shown by electrophoretic mobility shift assays but did not induce cell death. CONCLUSIONS: Fas and FasL are constitutively expressed in the myocardium and in cardiomyocytes. Myocardial expression of Fas is closely related to diastolic loading conditions in vivo. Signaling pathways emanating from Fas are coupled to an activation of the transcription factor AP-1 in cardiomyocytes.

Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis.

Here, we describe the isolation of adenine nucleotide translocase-1 (ANT-1) in a screen for dominant, apoptosis-inducing genes. ANT-1 is a component of the mitochondrial permeability transition complex, a protein aggregate connecting the inner with the outer mitochondrial membrane that has recently been implicated in apoptosis. ANT-1 expression led to all features of apoptosis, such as phenotypic alterations, collapse of the mitochondrial membrane potential, cytochrome c release, caspase activation, and DNA degradation. Both point mutations that impair ANT-1 in its known activity to transport ADP and ATP as well as the NH(2)-terminal half of the protein could still induce apoptosis. Interestingly, ANT-2, a highly homologous protein could not lead to cell death, demonstrating the specificity of the signal for apoptosis induction. In contrast to Bax, a proapoptotic Bcl-2 gene, ANT-1 was unable to elicit a form of cell death in yeast. This and the observed repression of apoptosis by the ANT-1-interacting protein cyclophilin D suggest that the suicidal effect of ANT-1 is mediated by specific protein-protein interactions within the permeability transition pore.

Metabolic effects of IGF-I in the growth retarded fetal sheep.

It has been shown that IGF-I has an anabolic effect in the normal fetus. However, there is evidence to suggest that there may be IGF-I resistance in the growth retarded fetus. Therefore, we investigated the effects of acute IGF-I infusion to chronically catheterised fetal sheep. At 128 days gestation, fetuses underwent a 4 h infusion of IGF-I (50 microg/kg/h). Three groups of animals were studied. Nine normally grown fetuses were studied as controls. Embolised animals (n=8) received microspheres into the uterine vasculature, and animals with spontaneous intra-uterine growth retardation (IUGR animals) (n=6) were fetuses found at post mortem to be spontaneously growth restricted. The effects of IGF-I infusion on feto-placental carbohydrate and protein metabolism were similar in our control group to previous similar experiments. IGF-I infusion decreased fetal blood glucose, oxygen, urea and amino-nitrogen concentrations, and inhibited placental lactate production. The same fetal blood metabolite concentrations also fell during IGF-I infusion in the embolised fetuses, but the effect on placental lactate production was not seen. The only effect of IGF-I infusion in the spontaneous IUGR animals was a fall in fetal blood amino-nitrogen concentrations. We conclude that fetal IGF-I infusion does not have the same anabolic effects in the growth retarded fetus as the normal fetus. In addition, the effects of IGF-I were different in the two growth retarded groups. Our data support previous evidence that the growth retarded fetus has altered IGF-I sensitivity, and this may vary depending on the cause, severity and duration of growth retardation.

P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death.

Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase-1, -3 and -8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP-induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP-induced cell death.

Fetal growth and placental function.

Fetal growth is largely determined by the availability of nutrients to the fetus. The fetus is at the end of a supply line that ensures delivery of nutrients from the maternal/uterine circulation to the fetus via the placenta. However, this supply line can not be regarded as a linear relationship. Maternal undernutrition will not only reduce global nutrient availability but will also influence the maternal and fetal somatotrophic axis. Both endocrine systems react in a very similar way to limited substrate supply. The hormones of the fetal somatotrophic axis, and in particular insulin-like growth factor (IGF)-1, are important regulators of fetal growth. Placental function is pivotal to materno-fetal nutrient and metabolite transfer. Placental function in turn, is heavily influenced by the maternal and fetal growth hormone (GH)-IGF-1 system. The placenta itself is also an active endocrine organ and it produces a large number of hormones including GH and IGF-1 as well their corresponding receptors. Thus the placenta can no longer be considered merely a passive conduit for fetal nutrition. Rather, it is actively involved in the integration of nutritional and endocrine signals from the maternal and fetal somatotrophic axes.

Oxidative stress and hypoxia/reoxygenation trigger CD95 (APO-1/Fas) ligand expression in microglial cells.

Apoptosis plays an important role in neurodegeneration, although the mechanisms and mediators in the brain are largely unknown. Because microglial cells have been suggested to contribute to apoptosis in neurological disorders, we investigated the expression of the death ligand CD95L in this cell type. We found that, compared to classical mediators of microglial activation, the most potent inducer of CD95L was oxidative stress. Exposure of microglial cells to H2O2 or paraquat rapidly triggered CD95L mRNA and protein expression, associated with the activation of transcription factor NF-kappaB. Enhanced expression of CD95L was further found following exposure of cells to hypoxia and subsequent reoxygenation. Our results indicate a potential role of CD95L in oxidative stress-mediated cell death, ischemia/reperfusion and other diseases with a disturbed redox balance.

Role of reactive oxygen intermediates in activation-induced CD95 (APO-1/Fas) ligand expression.

Activation-induced cell death of T lymphocytes requires the inducible expression of CD95 (APO-1/Fas) ligand, which triggers apoptosis in CD95-bearing target cells by an autocrine or paracrine mechanism. Although execution of the CD95 death pathway is largely independent of reactive oxygen intermediates, activation-induced cell death is blocked by a variety of antioxidants. In the present study, we investigated the involvement of redox processes in the regulation of CD95 ligand (CD95L) expression in Jurkat T cells. We show that various antioxidants potently inhibited the transcriptional activation of CD95L following T cell receptor ligation or stimulation of cells with phorbol ester and ionomycin. Conversely, a prooxidant such as hydrogen peroxide alone was able to increase CD95L expression. As detected by Western blot and cytotoxicity assays, functional expression of CD95L protein was likewise diminished by antioxidants. Inhibition of CD95L expression was associated with a decreased DNA binding activity of nuclear factor (NF)-kappaB, an important redox-controlled transcription factor. Moreover, inhibition of NF-kappaB activity by a transdominant IkappaB mutant attenuated CD95L expression. Our data suggest that, although reactive oxygen intermediates do not act as mediators in the execution phase of CD95-mediated apoptosis, they are involved in the transcriptional regulation of CD95L expression.

Antenatal therapy for intrauterine growth retardation.

Currently, there is no effective antenatal therapy for intrauterine growth retardation (IUGR). Although the IUGR fetus is undernourished in utero and there have been many attempts to treat IUGR with nutritional supplements, most studies have been poorly controlled, and there is no evidence to date that nutrient supplements can reverse the process of IUGR once it is established. Nutrient supplementation is also potentially risky and a combination of nutrients is likely to be needed. Alternative approaches to antenatal therapy for IUGR that show promise include fetal growth hormone and insulin-like growth factor I treatment to improve fetal growth. Fetal and maternal hormone supplements may also prove useful in IUGR by improving placental function and thus fetal substrate supply. Fetal enteral supplementation by the administration of growth factors and/or nutrients into the amniotic fluid may also prove effective and clinically feasible. It seems likely that combinations of these approaches will be required before effective therapy can be devised for the IUGR fetus in utero.

Activation of transcription factor NF-kappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways.

Mitogen-activated protein (MAP) kinases are important mediators of the cellular stress response. Here, we investigated the relationship between activation of the MAP kinase p38 and transcription factor NF-kappaB. Different forms of cellular stress were found to preferentially trigger either p38 or NF-kappaB. Arsenite or osmotic stress potently activated p38 but were ineffective in inducing NF-kappaB activation. Tumor necrosis factor-alpha and hydrogen peroxide, in contrast, led to NF-kappaB activation but only modestly stimulated p38. The activation of NF-kappaB was strongly abolished by antioxidants, while the activity of p38 and transcription factor AP-1 were increased. Inhibition of small GTPases including Rac and Cdc42 prevented p38 and AP-1 activation without interfering with NF-kappaB. In addition, inhibition of p38 by a pharmacological inhibitor or a dominant-negative mutant of MAP kinase kinase-6, an activator of the p38 pathway, interfered with NF-kappaB-dependent gene expression but not its DNA binding activity. Our results indicate that activation of p38 and NF-kappaB are mediated by separate pathways, which may converge further downstream in the cell nucleus. Different forms of cellular stress, however, initially trigger distinct signaling cascades involving either oxidative stress or GTPase-coupled pathways.

The Caenorhabditis elegans death protein Ced-4 contains a motif with similarity to the mammalian 'death effector domain'.

In the nematode Caenorhabditis elegans apoptosis is tightly regulated by a hierarchical set of genes. Two of these, ced-3 and ced-9, possess mammalian homologues encoding executional ICE proteases and inhibitory Bcl-2-related proteins, respectively. The function of a third key player, ced-4, is however completely unknown and no mammalian counterparts have been identified. Here we report that Ced-4 protein contains a structural region with similarity to the mammalian death effector domain which has previously been demonstrated to act as an important protein interaction motif in the signaling pathway of the mammalian surface receptor Fas (APO-1, CD95). Based on this finding and previously described genetic experiments, we propose that Ced-4, similar to the mammalian proteins FADD and FLICE, may possess a function as an adaptor protein in invertebrate apoptotic pathways.

Expression and regulation of cyclooxygenase-2 in rat microglia.

Increased levels of prostanoids have been implicated in various neuropathological diseases, although little is known about their cellular sources inside the brain. In this study, we analyzed the expression of cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, in rat microglia. COX-2 mRNA and protein as well as prostaglandin E2 formation were almost undetectable in unstimulated microglial cultures but were found to be strongly upregulated in response to lipopolysaccharide. However, in contrast to most peripheral cells, proinflammatory cytokines such as tumor necrosis factor alpha, interleukin-1 beta or interleukin-6 failed to markedly induce COX-2 expression. Similar effects were observed by analyzing transcription nuclear factor-kappa B (NF-kappa B) which was strongly activated in microglia by lipopolysaccharide but not by incubation with cytokines. Moreover, known inhibitors of NF-kappa B activation, such as dexamethasone and the antioxidant pyrrolidine dithiocarbamate, as well as the protein kinase C (PKC) inhibitor Gö6976, strongly reduced lipopolysaccharide-induced COX-2 transcription, indicating the involvement of NF-kappa B and PKC in COX-2 expression. Our results suggest that microglia may represent an important source of prostanoids in the brain, thus reinforcing their prominent role in cerebral inflammatory processes.

Oxidative stress and signal transduction.

Reactive oxygen intermediates (ROIs) are an evolutionarily ancient threat to all organisms. Both prokaryotic and higher eukaryotic cells are able to alter their genetic program in response to changes in the intracellular levels of ROIs. In bacteria and yeast, this response leads to the new synthesis of proteins that protect cells from the consequences of oxidative damage, such as DNA strand breaks, lipid peroxidation and oxidative damage of proteins. Intriguingly, higher organisms have also evolved cellular mechanisms to actively produce ROIs. There is increasing evidence that ROIs fulfil an important role as second messengers involved in signal transduction. We have proposed that ROIs have been conserved throughout evolution as universal pathogen messengers turning on genes with important functions in the immune response and cell proliferation. The higher eukaryotic transcription factors NF-kappa B and AP-1 will be described as proteins which are regulated by ROIs under a great variety of pathogenic conditions and initiate the new expression of genes with important roles in immune, inflammatory and other pathogen-related genetic responses.

Extracellular ATP activates transcription factor NF-kappaB through the P2Z purinoreceptor by selectively targeting NF-kappaB p65.

Cells of the macrophage lineage express a peculiar surface receptor for extracellular ATP, designated P2Z/P2X7 purinergic receptor, that induces pore formation and collapse of the plasma membrane potential. Although the function of the P2Z receptor is largely unknown, accumulating evidence implicates its role in cell signaling and immune reactions. Here, we investigated the effect of P2Z receptor ligation on the activation of NF-kappaB, a transcription factor controlling cytokine expression and apoptosis. Exposure of microglial cells to ATP but not other nucleotides resulted in potent NF-kappaB activation. This effect was specifically mediated by the P2Z receptor, because selective receptor antagonists prevented NF-kappaB activation. NF-kappaB activation required reactive oxygen intermediates and proteases of the caspase family, because it was abolished by antioxidants and specific protease inhibitors. The subunit composition of the ATP-induced NF- kappaB-DNA complex was rather unusual. Whereas exposure to LPS-induced prototypical NF-kappaB p50 homo- and p65 (RelA)/p50 heterodimers, ATP stimulation resulted in the sole appearance of a p65 homodimer. This is the first demonstration that a certain stimulus activates a particular NF-kappaB subunit. Because different NF-kappaB complexes exhibit distinct transcriptional and DNA-binding activities, ATP may control the expression of a subset of NF-kappaB target genes distinct from those activated by classical proinflammatory mediators.

Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis.

Among the many target genes of the transcription factor NF-kappaB are p53 and c-myc, both of which are involved in apoptosis. This prompted us to investigate the role of NF-kappaB in this process. We report that NF-kappaB is potently activated upon serum starvation, a condition leading to apoptosis in 293 cells. Similar to Bcl-2, a transdominant-negative mutant of the NF-kappaB p65 subunit partially inhibited apoptosis, indicating a direct involvement of the transcription factor in induction of cell death. As expected, the p65 mutant suppresses kappaB-dependent gene expression. Surprisingly, transiently or stably overexpressed Bcl-2 had the same effect. The transcription inhibitory activity of the two proteins correlated with their cell death protective potential. Like Bcl-2, the related protein Bcl-xL but not Bcl-xS was able to suppress kB-dependent transcription. Bcl-2 inhibited NF-kappaB activity by an unusual mechanism. It did not prevent the release of IkappaB in the cytoplasm but down-modulated the transactivating potential of nuclear p65. These data show that NF-kappaB can participate in apoptosis. We suggest that at least part of the anti-apoptotic potential of Bcl-2 may be explained from a hitherto undiscovered activity of Bcl-2 in controlling nuclear gene expression.

Pretreatment with bovine growth hormone is as effective as treatment during metabolic stress to reduce catabolism in fasted lambs.

The effects of recombinant bovine GH (rbGH) treatment on the insulin-like growth factor (IGF) axis and protein metabolism during fasting induced metabolic stress were evaluated in young lambs. To explore whether rbGH pretreatment alone might offer a degree of protection against nutritional stress, we compared the effects of rbGH given only before or during the fasting-induced metabolic stress with that given over the whole period. The animals were fed ad libitum for 5 days (well fed phase) and then fasted for 70 h (fasted phase). The rbGH was administered during either the well fed and the fasted phase (G-G), only during the well fed phase (G-S), or only during the fasted phase (S-G), and the effects were compared with those of saline treatment throughout both phases (S-S; n = 7/group). The rate of net protein catabolism, analyzed on the final day of the study, was reduced (P < 0.001) to a similar degree in all rbGH-treated groups compared with that in the S-S group. rbGH pretreatment was as effective as rbGH administered during the catabolic phase. Plasma IGF-I was increased (P < 0.001) in the well fed phase by rbGH treatment and decreased in the fasted phase in all groups. The rbGH treatment during the fasted phase resulted in a smaller fall in plasma IGF-I levels than saline treatment (P < 0.05, G-G vs G-S and S-G vs. S-S), but no difference was observed in the specific binding of [125I]ovine GH to the hepatic membranes from animals of the different groups. There was a negative correlation between net protein catabolism and plasma IGF-I levels (r = -0.48; P < 0.01) and specific binding of [125I]ovine GH to hepatic membranes (r = -0.56; P < 0.001). Plasma IGF-II levels were decreased by rbGH treatment during the well fed phase, but the responses to treatment during the fasted phase were variable, suggesting that plasma IGF-II is regulated in a different manner than plasma IGF-I. The fasting-induced fall (P < 0.05) in plasma concentrations of IGF-binding protein (IGFBP)-3 was reduced with rbGH treatment, and plasma concentrations IGFBP-2 were altered in an inverse manner. This study suggests that fasting-induced GH resistance can be alleviated by rbGH treatment independent of whether treatment is commenced before or after the onset of catabolic stress. Our observation of prolonged anticatabolic action of prophylactic rbGH treatment supports the proposal that prophylactic use of GH may reduce the degree of catabolism associated with subsequent interventions and, thus, improve clinical outcome.