Dose of Trp53, the main keeper of genome stability, influences tumorigenesis; however, the causes underlying and driving tumorigenesis over time by the loss of a single p53 allele are still poorly characterized. Here, we found that single p53 allele loss specifically impacted the oxidative, DNA damage and inflammatory status of hematopoietic lineages. In particular, single Trp53 allele loss in mice triggered oxidative stress in peripheral blood granulocytes and spleenocytes, whereas lack of two Trp53 alleles produced enhanced oxidative stress in thymus cells, resulting in a higher incidence of lymphomas in the Trp53 knockout (KO) mice compared with hemizygous (HEM). In addition, single or complete loss of Trp53 alleles, as well as p53 downregulation, led to a differential increase in basal, LPS- and UVB-induced expression of a plethora of pro-inflammatory cytokine, such as interleukin-12 (Il-12a), TNFα (Tnfa) and interleukin (Il-23a) in bone marrow-derived macrophage cells (BMDMs) compared to WT cells. Interestingly, p53-dependent increased inflammatory gene expression correlated with deregulated expression of the NF-κB pathway inhibitor IκBα. Chromatin immunoprecipitation data revealed decreased p65 binding to Nfkbia in the absence of p53 and p53 binding to Nfkbia promoter, uncovering a novel crosstalk mechanism between p53 and NF-κB transcription factors. Overall, our data suggest that single Trp53 allele loss can drive a sustained inflammatory, DNA damage and oxidative stress response that, over time, facilitate and support carcinogenesis.

Brown adipose tissue (BAT) plays a central role in the regulation of whole-body energy and glucose homeostasis owing to its elevated capacity for lipid and glucose oxidation. The BAT thermogenic function, which is essential for the defense of body temperature against exposure to low environmental temperatures, relies on the expression in the inner membrane of brown adipocyte's mitochondria of uncoupling protein-1, a protein that uncouples substrate oxidation from oxidative phosphorylation and leads to the production of heat instead of ATP. BAT thermogenesis depends on proper mitochondrial biogenesis during the differentiation of brown adipocytes. Despite the data that support a role for Endonuclease G (EndoG) in the process of mitochondrial biogenesis, its function in BAT has not been explored. Here, using an EndoG knockout mouse model, we demonstrate that EndoG is not essential for the expression of mitochondrial genes involved in substrate oxidation or for the induction of thermogenic genes in BAT in response to cold exposure. We also show that a lack of EndoG is associated with an increased expression of thermogenic genes (ie, uncoupling protein-1, peroxisome proliferator-activated receptor-γ coactivator-1α) in white adipose tissue (WAT) that correlates with the appearance of brown adipocyte-like cells interspersed among white adipocytes. Interestingly, the increased browning of WAT elicited by the lack of EndoG was associated with a better glucose tolerance and reduced fat mass. Our results suggest that the induction of browning in WAT by means of inhibiting EndoG activity appears as a potential therapeutic strategy to prevent obesity and ameliorate glucose intolerance.

Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart’s cellularity and maturation during development, contributing novel information about caspase biology and heart development.

TREX2 is a 3'-DNA exonuclease specifically expressed in keratinocytes. Here, we investigated the relevance and mechanisms of TREX2 in ultraviolet (UV)-induced skin carcinogenesis. TREX2 expression was up-regulated by chronic UV exposure whereas it was de-regulated or lost in human squamous cell carcinomas (SCCs). Moreover, we identified SNPs in the TREX2 gene that were more frequent in patients with head and neck SCCs than in healthy individuals. In mice, TREX2 deficiency led to enhanced susceptibility to UVB-induced skin carcinogenesis which was preceded by aberrant DNA damage removal and degradation as well as reduced inflammation. Specifically, TREX2 loss diminished the up-regulation of IL12 and IFN gamma, key cytokines related to DNA repair and antitumor immunity. In UV-treated keratinocytes, TREX2 promoted DNA repair and passage to late apoptotic stages. Notably, TREX2 was recruited to low-density nuclear chromatin and micronuclei, where it interacted with phosphorylated H2AX histone, which is a critical player in both DNA repair and cell death. Altogether, our data provide new insights in the molecular mechanisms of TREX2 activity and establish cell autonomous and non-cell autonomous functions of TREX2 in the UVB-induced skin response.

Tissue repair is a complex process, which may be favoured or inhibited by different factors. Potassium apigenin (AP) and other flavonoids present in verbena extract (PLX(®) ) possess powerful anti-inflammatory properties. The aim of this study was to evaluate the effects of topical treatment with AP and PLX gels on wounds inflicted on SKH-1/CRL mice. Forty-eight SKH-1 mice were used (4 groups with 12 animals each), which were subjected to wound excision with a round scalpel, 4 mm in diameter, on the dorsal skin. The animals were divided into four groups: Group I received topical applications of apigenin gel; Group II received PLX gel; Group III received vehicle gel; Group IV acted as control. Wound contraction, reepithelialisation, inflammation and neovascularisation (by means of immunohistochemical staining with anti-laminin) were recorded at study periods established at 2, 7 and 14 days. Reepithelialisation was faster in Groups I and II at 7 days (56·25% grade 3 and 43·75% grade 4) compared with the other groups. The degree of inflammation showed improvement with a tendency towards statistical significance in Groups I and II at 2 and 7 days. Anti-laminin staining was more intense in the group treated with PLX at the 2- and 7-day periods. Topical treatment with PLX gel improved the degree of reepithelialisation and inflammation, and favoured neo-vascularisation of the wounds at 2 and 7 days following surgery.

Endonuclease G (EndoG) has been largely related with a role in the modulation of a caspase-independent cell death pathway in many cellular systems. However, whether this protein plays a specific role in the brain remains to be elucidated. Here we have characterized the behavioral phenotype of EndoG(-/-) null mice and the expression of the nuclease among brain regions. EndoG(-/-) mice showed normal neurological function, learning, motor coordination and spontaneous behaviors. However, these animals displayed lower activity in a running wheel and, strikingly, they were consistently less anxious compared to EndoG(+/+) mice in different tests for anxiety such as plus maze and dark-light test. We next evaluated the expression of EndoG in different brain regions of wild type mice and found that it was expressed in all over but specially enriched in the striatum. Further, subcellular biochemical experiments in neocortical samples from wild type mice revealed that EndoG is localized in pre-synaptic compartments but not in post-synaptic compartments. Altogether these findings suggest that EndoG could play a highly specific role in the regulation of anxiety by modulating synaptic components.

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-xL. These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells.

d -Fagomine is an iminosugar originally isolated from seeds of buckwheat ( Fagopyrum sculentum Moench), present in the human diet and now available as a pure crystalline product. We tested d -fagomine for activities connected to a reduction in the risk of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. The activities were: intestinal sucrase inhibition in vitro (rat mucosa and everted intestine sleeves), modulation of postprandial blood glucose in rats, bacterial agglutination and bacterial adhesion to pig intestinal mucosa. When ingested together with sucrose or starch, d -fagomine lowered blood glucose in a dose-dependent manner without stimulating insulin secretion. d -Fagomine reduced the area under the curve (0–120 min) by 20 % ( P < 0·01) and shifted the time to maximum blood glucose concentration ( T max ) by 15 min at doses of 1–2 mg/kg body weight when administered together with 1 g sucrose/kg body weight. Moreover, d -fagomine (0·14 m m ) agglutinated 60 % of Enterobacteriaceae ( Escherichia coli , Salmonella enterica serovar Typhimurium) populations ( P < 0·01), while it did not show this effect on Bifidobacterium spp. or Lactobacillus spp. At the same concentration, d -fagomine significantly ( P < 0·001) inhibited the adhesion of Enterobacteriaceae (95–99 % cells in the supernatant) and promoted the adhesion of Lactobacillus acidophilus (56 % cells in the supernatant) to intestinal mucosa. d -Fagomine did not show any effect on bacterial cell viability. Based on all this evidence, d -fagomine may be used as a dietary ingredient or functional food component to reduce the health risks associated with an excessive intake of fast-digestible carbohydrates, or an excess of potentially pathogenic bacteria.

Elevated left ventricular mass, a highly heritable trait, is an important risk factor for heart failure and death. Stuart Cook and colleagues genetically dissect a locus in the rat associated with blood-pressure-independent cardiac hypertrophy and identify endonuclease G (ENDOG) as a key regulator of hypertrophy at this locus. They further show that the Endog gene is involved in proper mitochondrial function and is modulated by ERR-α and PGC1α, master regulators of mitochondrial and cardiac function. Loss of function of ENDOG causes impaired mitochondrial respiration and increased production of reactive oxygen species, which may contribute to the observed cardiac hypertrophy. Left ventricular mass (LVM) is a highly heritable trait1 and an independent risk factor for all-cause mortality2. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation3, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood4,5. Unbiased systems genetics approaches in the rat6,7 now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis8 but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function)9,10,11, interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin‐positive (PV + ) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV + interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

It is well documented that schizophrenia patients exhibit dysfunction in various cognitive domains, including attention/vigilance, as demonstrated by impaired performance in the myriad of Continuous Performance Tests (CPTs). NMDA receptor antagonists provide a pharmacological model in animals of the cognitive disruption presented in the disorder. We therefore examined the effects of a sub-chronic PCP treatment regimen (5.0 mg/kg 7-days bi-daily) in the recently developed rodent test of vigilance, the 5-Choice Continuous Performance Test (5C-CPT). We assessed the effects of this regimen after at least a 7-day washout period on both baseline performance and when the attentional load was increased. Sub-chronic PCP treatment impaired 5C-CPT performance in a manner consistent with impaired vigilance in patients with schizophrenia, with reduced hit rate and impaired signal sensitivity. These effects were only evident when performance was challenged following parameter manipulations. These data demonstrate that attention/vigilance is sensitive to disruption following sub-chronic PCP treatment in a pre-clinical task that may demonstrate increased analogy to human vigilance tasks. Although the PCP-induced attentional deficits are not as large as those deficits observed in other domains, these data provide evidence that this pharmacological model can affect multiple cognitive domains and may be useful for assessing putative pro-cognitive therapeutics for schizophrenia.This article is part of a Special Issue entitled ‘Schizophrenia’.

It has been demonstrated that proinsulin C-peptide possesses several biological activities and that its specific binding sites are present on the surface of cell membranes. However, the molecular and cellular mechanisms of C-peptide actions are poorly known. In the present study we examined the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in C-peptide effects. C-peptide induced the phosphorylation of MAPK [p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2] in Swiss 3T3 and 3T3-F442A fibroblasts but not in 3T3-L1 fibroblasts and some other cell lines such as L6E9 muscle cells. In Swiss 3T3 cells, C-peptide-induced phosphorylation of MAPK was dependent on time and concentration, being maximal at 1min and at 1nM C-peptide and was accompanied by an increase in MAPK activity and MAPK kinase (MEK) phosphorylation. The MAPK phosphorylation by C-peptide was abolished by treatment with pertussis toxin (PTX) and also with a MEK inhibitor, PD 98059. In addition, MAPK phosphorylation was attenuated by treatment with a phosphoinositide 3-kinase (PI-3K) inhibitor, wortmannin, and with a protein kinase C (PKC) inhibitor, GF109203X, and by down-regulation of PKC by prolonged treatment with PMA. Similar effects of the inhibitors and PTX were found on the MAPK phosphorylation induced by neuropeptide Y. These results suggest that C-peptide activates MAPK through a putative Gi/Go-linked receptor for C-peptide and through PI-3K-dependent and PKC-dependent pathways.

Aims/hypothesis. To investigate whether proinsulin C peptide influences sympathetic nerve activity directly or indirectly through parasympathetic nerve activity. Methods. The proliferative response of splenic lymphocytes to Concanavalin A (ConA response) which is known to be suppressed by subjection of rats to footshock or intracerebroventricular injection of corticotropin-releasing factor through sympathetic nerve activation was measured. Effect of C peptide alone or before subjection to footshock or injection of corticotropin-releasing factor was examined. Results. Intraperitoneal injection of C peptide was without effect on the basal ConA response, while subjection to footshock or injection of corticotropin-releasing factor lowered it. In contrast, prior injection of C peptide obviated the footshock and corticotropin-releasing factor-induced suppression of the response. When given intracerebroventricularly, C peptide was also effective at much smaller doses. Prior injection of atropine cancelled the C-peptide effects. Conclusion/interpretation. Our results indicate that C peptide counteracts the sympathetic nerve-mediated suppression of splenic lymphocyte proliferation in an atropine-sensitive manner. Thus, C peptide probably activates the parasympathetic nervous system through the afferent mechanism, that in turn antagonizes the sympathetic nerve-mediated suppression of splenic lymphocyte functions. [Diabetologia (2000) 43: 1512–1517]

Brown adipose tissue (BAT) is the specific site for metabolic heat production in mammals. To establish a novel immortal brown adipocyte cell line, the stromal-vascular fraction containing preadipocytes was obtained from interscapular BAT of mice deficient of a tumor-suppressor genep53.Thep53-deficient cells, tentatively named as HB2 cells, could be culturedin vitroafter repeated passages and differentiated into adipocytes in the presence of insulin, T3and/or troglitazone, expressing some adipocyte-specific genes and accumulating intracellular lipid droplets. The mRNA level of uncoupling protein 1 (UCP1), a mitochondrial protein specifically present in brown adipocytes, was undetectable in HB2 preadipocytes, but increased after adipose differentiation. In HB2 adipocytes, UCP1 mRNA expression was markedly activated after stimulation of the β-adrenergic receptor pathway. The mRNA of UCP2 and UCP3, recently cloned isoforms of UCP1, were also detected in HB2 adipocytes, but their levels were not influenced by adrenergic stimulation. Thus HB2 cells seem useful forin vitrostudies of BAT and UCP functions.

Uncoupling protein 2 (UCP2) mRNA expression and function was examined in rat primary cultured hepatocytes. UCP2 mRNA was not expressed in freshly isolated hepatocytes, but appeared during a 24–144 h primary culture period. Isolated mitochondria from 144 h cultured hepatocytes showed a lower oxygen consumption rate in the presence of succinate and ADP. However, the ratio of the oxygen consumption rate when media contained succinate alone to that with succinate and ADP was increased by 166% versus control mitochondria. Moreover, the mitochondrial potential in the presence of succinate was decreased by 60%, indicating the potential role of UCP2 in hepatocyte mitochondria as an active uncoupler.

Uncoupling protein 3 (UCP3), expressed abundantly in the skeletal muscle, is one of the carrier proteins dissipating the transmitochondrial electrochemical gradient as heat, and thereby has been implicated in the regulation of energy metabolism. We have investigated UCP3 mRNA expression in the widely used L6 myocyte cell line by Northern blot analysis. UCP3 mRNA was not detected in L6 myoblasts, but appeared after their differentiation to myotubes. The UCP3 mRNA level was increased when L6 myotubes were treated with increasing concentrations of triiodothyronine (T3), oleic acid, α‐bromopalmitate and carbacyclin, a non‐selective ligand of peroxisome proliferator‐activated receptors (PPARs), whereas it was not influenced when treated with selective ligands of PPARα (WY 14 643) and PPARγ (troglitazone). A ligand of retinoid X receptor (RXR), 9‐cis retinoic acid, was also effective by itself and in combination with carbacyclin in stimulating UCP3 mRNA expression. The mRNA analysis of individual PPAR isoforms revealed that L6 cell expressed a significant level of PPARδ but undetectable levels of PPARα and PPARγ. These results suggest that UCP3 expression in myocytes is differentiation‐dependent and regulated by the T3 receptor, RXR and PPARδ.

OBJECTIVE: To determine the effect of 3-hydroxybutyrate (3OHB) on the thermogenic response to noradrenaline (NA) in lean and genetically obese Zucker fa/fa rats. DESIGN: Rats were infused with 18.7 nmol·kg −1 ·min −1 of NA, supplemented, for 15 min, with 66.7 μmol·kg −1 ·min −1 of R-3-hydroxybutyrate (3OHB). SUBJECTS: Pentobarbital-anaesthetized lean and obese Zucker rats. MEASUREMENTS: Aortic and interscapular brown adipose tissue (BAT) temperature; plasma NA, 3OHB, glucose and insulin levels during infusion. RESULTS: The NA-induced increase in aortic and BAT temperature was more marked in lean than in obese rats. In lean rats, the rise was arrested by 3OHB; but in obese rats 3OHB had no effect. Infusion of saline, glucose or 3OHB in the absence of NA did not induce changes in either temperature. NA infusion resulted in a rapid increase in plasma NA to 45–50 nM in both groups; this plateau was maintained for up to 60 min. The presence of 3OHB decreased the plasma NA of lean rats, but did not affect the plasma NA of the obese rats. Blood 3OHB rose to 1.2 mM during 3OHB infusion in both groups, and decreased on cessation of infusion. Blood glucose levels increased with NA infusion in both groups; the presence of high 3OHB levels decreased glucose levels only in lean rats. CONCLUSION: The changes in NA levels induced by 3OHB may help explain the effects observed on temperature and glucose. The defective thermogenic system of obese rats cannot be modulated by 3OHB, unlike thermogenesis in lean rats, on which 3OHB has a marked effect.

ABSTRACT Pentobarbital-anaesthetized male Wistar rats were infused with 6 μg kg−1 min−1 of noradrenaline. The infusion was supplemented with 8.5 mg kg−1 min−1 of D-3-hydroxybutyrate (3-OHB) for 15 min in order to determine its effect on the adrenergic response of the rat. Plasma levels of noradrenaline rose to a plateau of approximately 50 nmol l−1 with infusion. In the group infused with noradrenaline alone, noradrenaline levels were maintained for 1 h. Supplementation with 3-OHB induced a decrease in plasma noradrenaline level that was inversely correlated with 3-OHB level. Aortic and interscapular brown adipose tissue temperatures increased with noradrenaline infusion, but the rise was arrested by 3-OHB; replacing 3-OHB with glucose had no effect. Infusion of saline, glucose or 3-OHB in the absence of noradrenaline did not induce a rise in temperature in either tissue. Blood 3-OHB concentration increased to 1.2 mmol l−1 during 3-OHB infusion, decreasing rapidly at the end of infusion. Blood glucose levels increased with noradrenaline infusion; the presence of high 3-OHB levels decreased glucose concentration. The effects observed were transient and dependent on 3-OHB concentration; these effects may help explain most of the other effects of noradrenaline described here. The role of 3-OHB as a regulator of adrenergic responses seems to be part of a complex fail-safe mechanism which prevents wasting.

OBJECTIVE To determine whether adipose tissue removes a significant proportion of circulating insulin in the obese rat in vivo. DESIGN Blood samples were obtained from the carotid artery and the venous side of hind leg muscle, epididymal fat pads and liver (portal and suprahepatic veins), they were used for estimation of insulin disappearance across the tissue. SUBJECTS Anaesthetized Zucker lean (Fa/?) and obese (fa/fa) adult male rats. MEASUREMENTS Blood flow, glycaemia and insulinaemia. RESULTS In lean rats, the liver accounted for practically all insulin extraction (498 +/- 149 fmol.s-1), removing more than half of the portal insulin in a single pass. In the obese rats, the liver extracted a similar amount in absolute terms (397 +/- 108 fmol.s-1), representing only 16% of portal insulin. In the hind leg muscle of both groups and in the adipose tissue of lean rats, insulin balances were not different from zero; but in obese rats, adipose tissue extracted a similar amount of insulin (405 +/- 127 fmol.s-1) to that removed by the liver. CONCLUSIONS In the Zucker obese rat, peripheral adipose tissue removes as much insulin from the blood as the liver does, in contrast with lean rats, in which the latter is responsible for practically all insulin extraction.