When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.

Reducing or eliminating expression of a given gene is likely to require multiple methods to ensure coverage of all of the genes in a given mammalian cell. We and others [Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C., and Baker, C. C. (1994) Mol. Cell. Biol. 14, 5278 -5289] have previously shown that U1 small nuclear (sn) RNA, both natural or with 5 end mutations, can specifically inhibit reporter gene expression in mammalian cells. This inhibition occurs when the U1 snRNA 5 end base pairs near the polyadenylation signal of the reporter gene's pre-mRNA. This base pairing inhibits poly(A) tail addition, a key, nearly universal step in mRNA biosynthesis, resulting in degradation of the mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by transiently or stably expressed 5 end-mutated U1 snRNA. Also, we determine the inhibitory mechanism and establish a set of rules to use this technique and to improve the efficiency of inhibition. Two U1 snRNAs base paired to a single pre-mRNA act synergistically, resulting in up to 700-fold inhibition of the expression of specific reporter genes and 25-fold inhibition of endogenous genes. Surprisingly, distance from the U1 snRNA binding site to the poly(A) signal is not critical for inhibition, instead the U1 snRNA must be targeted to the terminal exon of the pre-mRNA. This could reflect a disruption by the 5 end-mutated U1 snRNA of the definition of the terminal exon as described by the exon definition model. mutant U1 snRNAs ͉ gene expression inhibition ͉ polyadenylation inhibition

Most of the genes induced by hypoxia are regulated by a family of transcription factors termed hypoxia-inducible factors (HIF). Under normoxic conditions, HIF␣proteins are very unstable due to hydroxylation by a recently described family of proline hydroxylases termed EGL-Nine homologs (EGLN). Upon hydroxylation, HIF␣ is recognized by the product of the tumor suppressor vhl and targeted for proteosomal degradation. Since EGLNs require oxygen to catalize HIF hydroxylation, this reaction does not efficiently occur under low oxygen tension. Thus, under hypoxia, HIF␣ escapes from degradation and transcribes target genes. The mRNA levels of two of the three EGLNs described to date are induced by hypoxia, suggesting that they might be novel HIF target genes; however, no proof for this hypothesis has been reported. Here we show that the induction of EGLN1 and -3 by hypoxia is found in a wide range of cell types. The basal levels of EGLN3 are always well below those of EGLN1 and EGLN2, and its induction by hypoxia is larger than that found for EGLN1. The inhibitor of transcription, actinomycin D, prevents the increase of EGLN3 mRNA induced by hypoxia, indicating that it is due to enhanced gene expression. Interestingly, EGLN1 and EGLN3 mRNAs were also triggered by EGLN inhibitors, suggesting the involvement of HIF␣ in the control of its transcription. In agreement with this possibility, pVHL-deficient cell lines, which present high HIF activity under normoxia, also showed dramatically increased normoxic levels of EGLN3. Moreover, the overexpression of an oxygen-insensitive mutant form of HIF␣ resulted in increased normoxic levels of EGLN3 mRNA. Finally, hypoxic induction of EGLNs was not observed in cells lacking functional HIF␣.Mammalian cells are critically dependent on oxygen for survival; thus, it is not surprising that an evolutionarily conserved machinery able to sense oxygen levels is present in almost every cell type studied. The activity of a variety of signaling molecules has been found to be altered in response to changes in oxygen tension, including ion channels (1), kinases (2-7), and transcription factors (2, 8 -13). Among them, the most common and best understood response is the activation of a group of basic helix-loop-helix-Per/Aryl hydrocarbon receptor translocator/Sim domain transcription factors, called hypoxiainducible factors (HIF) 1 (14). In fact, HIF is responsible for the induction of the vast majority of genes up-regulated in response to decreased oxygen tension. HIF transcription factors are heterodimers of a constitutively expressed ␤ subunit (HIF-␤; also known as the aryl hydrocarbon receptor translocator) and an oxygen-regulated ␣ subunit (HIF-␣). Although there are three types of ␣ subunits, HIF-1␣, HIF-2␣ (also known as the endothelial Per/Aryl hydrocarbon receptor translocator/Sim domain), and HIF-3␣, encoded by different genes, it is currently accepted that they are regulated in a similar fashion by oxygen availability. The expression of HIF␤ and ␣ subunits genes, their mRNA stability, ...

Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing beta-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed beta-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.

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Pneumoperitoneum in children may be due to causes that do not require urgent surgery (cardiopulmonary resuscitation manoeuvres, severe respiratory pathology or mechanical ventilation). Surgery in these cases could even worsen the prognosis. We present the case of a male infant, ex-preterm, with a history of necrotizing enterocolitis and ileal perforation at birth, requiring laparotomy and intestinal resection on two occasions and developing a secondary microcolon, due to disuse. At six months, after transitioning to full oral feeding, he presented abdominal distension with extensive intestinal pneumatosis and pneumoperitoneum on radiographs. His general appearance was good with normal intestinal transit and no peritonitis. The patient remained fasting with intravenous antibiotics, nasogastric decompression and parenteral nutrition. The evolution was favourable with oral feeding restarting on the seventh day of admission. The existence of pneumoperitoneum does not always require a laparotomy and global assessment of the patient by an interdisciplinary health team may avoid unnecessary aggressive treatments.

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