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Synchronization to periodic cues such as food/water availability and light/dark cycles is crucial for living organisms' homeostasis. Both factors have been heavily influenced by human activity, with artificial light at night (ALAN) being an evolutionary challenge imposed over roughly the last century. Evidence from studies in humans and animal models shows that overt circadian misalignment, such as that imposed to about 20% of the workforce by night shift work (NSW), negatively impinges on the internal temporal order of endocrinology, physiology, metabolism, and behavior. Moreover, NSW is often associated to mistimed feeding, with both unnatural behaviors being known to increase the risk of chronic diseases, such as eating disorders, overweight, obesity, cardiovascular, metabolic (particularly type 2 diabetes mellitus) and gastrointestinal disorders, some types of cancer, as well as mental disease including sleep disturbances, cognitive disorders, and depression. Regarding deleterious effects of ALAN on reproduction, increased risk of miscarriage, preterm delivery and low birth weight have been reported in shift-worker women. These mounting lines of evidence prompt further efforts to advance our understanding of the effects of long-term NSW on health. Emerging data suggest that NSW with or without mistimed feeding modify gene expression and functional readouts in different tissues/organs, which seem to translate into persistent cardiometabolic and endocrine dysfunction. However, this research avenue still faces multiple challenges, such as functional characterization of new experimental models more closely resembling human long-term NSW and mistimed feeding in males versus females; studying further target organs; identifying molecular changes by means of deep multi-omics analyses; and exploring biomarkers of NSW with translational medicine potential. Using high-throughput and systems biology is a relatively new approach to study NSW, aimed to generate experiments addressing new biological factors, pathways, and mechanisms, going beyond the boundaries of the circadian clock molecular machinery.

Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A3 Adenosine Receptor (A3AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A3AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A3AR on cell migration/invasion was evaluated using the A3AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A3AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAP-dependent activation of A3AR under hypoxia.

Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease-modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here we studied the impact of IGF2 signaling on protein aggregation in models of Huntington’s disease (HD) as proof-of-concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 were independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human post-mortem brain tissue, and blood samples from HD patients showed a reduction of IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein aggregates.One sentence summaryIGF2 reduces the load of intracellular protein aggregates through the extracellular disposal of the mutant protein.

Key points Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Here we explored the developmental impact of gestational chronodisruption (chronic photoperiod shift, CPS) on adult and fetal adrenal biorhythms and function. We found that gestational chronodisruption altered fetal and adult adrenal function, at the molecular, morphological and physiological levels. The differences between control and CPS offspring suggest desynchronization of the adrenal circadian clock and steroidogenic pathway, leading to abnormal stress responses and metabolic adaptation, potentially increasing the risk of developing chronic diseases. Abstract Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Indeed, an abnormal photoperiod during gestation alters fetal development, inducing long-term effects on the offspring. Accordingly, we carried out a longitudinal study in rats, exploring the impact of gestational chronodisruption on the adrenal biorhythms and function of the offspring. Adult rats (90 days old) gestated under chronic photoperiod shift (CPS) decrease the time spent in the open arm zone of an elevated plus maze to 62% and increase the rearing time to 170%. CPS adults maintained individual daily changes in corticosterone, but their acrophases were distributed from 12.00 h to 06.00 h. CPS offspring maintained clock gene expression and oscillation, nevertheless no daily rhythm was observed in genes involved in the regulation and synthesis of steroids. Consistent with adult adrenal gland being programmed during fetal life, blunted daily rhythms of corticosterone, core clock gene machinery, and steroidogenic genes were observed in CPS fetal adrenal glands. Comparisons of the global transcriptome of CPS versus control fetal adrenal gland revealed that 1078 genes were differentially expressed (641 down-regulated and 437 up-regulated). In silico analysis revealed significant changes in Lipid Metabolism, Small Molecule Biochemistry, Cellular Development and the Inflammatory Response pathway (z score: 48-20). Altogether, the present results demonstrate that gestational chronodisruption changed fetal and adult adrenal function. This could translate to long-term abnormal stress responses and metabolic adaptation, increasing the risk of developing chronic diseases.

Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A

Recent reports account for altered metabolism in adult offspring from pregnancy subjected to abnormal photoperiod, suggesting fetal programming of liver physiology. To generate a pipeline of subsequent mechanistic experiments addressing strong candidate genes, here we investigated the effects of constant gestational light on the fetal liver transcriptome. At 10 days of gestation, dams were randomized in two groups ( n = 7 each): constant light (LL) and normal photoperiod (12 h light/12 h dark; LD). At 18 days of gestation, RNA was isolated from the fetal liver and subjected to DNA microarray (Affymetrix platform for 28,000 genes). Selected differential mRNAs were validated by quantitative PCR (qPCR), while integrated transcriptional changes were analyzed with Ingenuity Pathway Analysis and other bioinformatics tools. Comparison of LL relative to LD fetal liver led to the following findings. Significant differential expression was found for 3,431 transcripts (1,960 upregulated and 1,471 downregulated), with 393 of them displaying ≥ 1.5-fold change. We validated 27 selected transcripts by qPCR, which displayed fold-change values highly correlated with microarray ( r2= 0.91). Different markers of nonalcoholic fatty liver disease were either upregulated (e.g., Ndn and Pnpla3) or downregulated (e.g., Gnmt, Bhmt1/2, Sult1a1, Mpo, and Mat1a). Diverse pathways were altered, including hematopoiesis, coagulation cascade, complement system, and carbohydrate and lipid metabolism. The microRNAs 7a-1, 431, 146a, and 153 were upregulated, while the abundant hepatic miRNA 122 was downregulated. Constant gestational light induced extensive modification of the fetal liver transcriptome. A number of differentially expressed transcripts belong to fundamental functional pathways, potentially contributing to long-term liver disease.

Chronic exposure to light at night, as in shift work, alters biological clocks (chronodisruption), negatively impacting pregnancy outcome in humans. Actually the interaction of maternal and fetal circadian systems could be a key factor determining a fitting health in adults. We propose that chronic photoperiod shift (CPS) during pregnancy alter maternal circadian rhythms and impair circadian physiology in the adult offspring, increasing health risks. Pregnant rats were exposed to normal photoperiod (12 h light, 12 h dark) or to CPS until 85% of gestation. The effects of gestational CPS were evaluated on the mother and adult offspring. In the mother we measured rhythms of heart rate, body temperature, and activity through gestation and daily rhythms of plasma variables (melatonin, corticosterone, aldosterone, and markers of renal function) at 18 days of gestation. In adult offspring, we measured rhythms of the clock gene expression in the suprachiasmatic nucleus (SCN), locomotor activity, body temperature, heart rate, blood pressure, plasma variables, glucose tolerance, and corticosterone response to ACTH. CPS altered all maternal circadian rhythms, lengthened gestation, and increased newborn weight. The adult CPS offspring presented normal rhythms of clock gene expression in the SCN, locomotor activity, and body temperature. However, the daily rhythm of plasma melatonin was absent, and corticosterone, aldosterone, renal markers, blood pressure, and heart rate rhythms were altered. Moreover, CPS offspring presented decreased glucose tolerance and an abnormal corticosterone response to ACTH. Altogether these data show that gestational CPS induced long-term effects on the offspring circadian system, wherein a normal SCN coexists with altered endocrine, cardiovascular, and metabolic function.

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT-PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose-1,6-bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co-localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 μM Fru-1,6-P2. These data indicate the presence of a modulated enzyme–enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis. © 2004 Wiley-Liss, Inc.

article i nfo Cardiac gene networks Whole transcriptome Gestational chronodisruption Fetal programming of adult disease Left ventricle hypertrophy We recently reported that gestational chronodisruption induces fetal growth restriction and marked effects on fetal adrenal physiology. Here, whole-transcriptome profiling was used to test whether gestational chronodisruption modifies gene expression in the fetal heart, potentially altering cardiac development. At day 10 of gestation (E10), pregnant rats were randomized in two groups: constant light (LL) and control 12 h light/12 h dark photope- riod (LD). RNAisolated from E18 heartwas subjectedto microarray analysis(Affymetrix platform for28,000 genes). Integrated transcriptional changes were assesse db y gene ontology and pathway analysis. Signi ficant differential expression was found for 383 transcripts in LL relative to LD fetal heart (280 up-regulated and 103 down- regulated); with 42 of them displaying a 1.5-fold or greater change in gene expression. Deregulated markers of cardiovascular disease accounted for alteration of diverse gene networks in LL fetal heart, including local steroido- genesis and vascular calcification, as well as cardiac hypertrophy, stenosis and necrosis/cell death. DNA integrity was also overrepresented, including a 2.1-fold increase of Hmga1 mRNA, which encodes for a profuse architectural transcription factor. microRNA analysis revealed up-regulation of miRNAs 218-1 and 501 and concurrent down- regulation of their validated target genes. In addition, persistent down-regulation of Kcnip2 mRNA and hypertrophy of the left ventricle were found in the heart from 90 days-old offspring from LL mothers. The dysregulation of a rel- evant fraction of the fetal cardiac transcriptome, together with the diversity and complexity of the gene networks altered by gestational chronodisruption, suggest enduring molecular changes which may shape the hypertrophy observed in the left ventricle of adult LL offspring.

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of clock genes and clock-controlled genes as well as it lowered the content and precluded the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night; supporting a role of melatonin on overall fetal development and pointing to it as a 'time giver' for the fetal adrenal gland. Thus, the present results collectively support that the maternal circadian rhythm of melatonin is a key signal for the generation and/or synchronization of the circadian rhythms in the fetal adrenal gland. In turn, low levels and lack of a circadian rhythm of fetal corticosterone may be responsible of fetal growth restriction; potentially inducing long term effects in the offspring, possibility that warrants further research.

Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In contrast, here we demonstrate that liver fructose-1,6-bisphosphatase (FBPase) is highly expressed both in human and rat pancreas. Interestingly, pancreatic FBPase is active and functional, and is inhibited by AMP and fructose-2,6-bisphosphate (Fru-2,6-P2). These results suggest that FBPase may participate as a component of a metabolic sensing mechanism present in the pancreas. Immunolocalization analysis showed that FBPase is expressed both in human and rat Langerhans islets, specifically in β cells. In humans, FBPase was also located in the canaliculus and acinar cells. These results indicate that FBPase coupled with phosphofructokinase (PFK) plays a crucial role in the metabolism of pancreatic islet cells. The demonstration of gluconeogenic recycling of trioses as a new metabolic signaling pathway may contribute to our understanding of the differences between the insulin secretagogues trioses, fructose, and glucose in pancreas. © 2005 Wiley-Liss, Inc.

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT-PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose-1,6-bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co-localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 microM Fru-1,6-P2. These data indicate the presence of a modulated enzyme-enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.