REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

Cetoacidosis inducida por corticoides y gatifloxacina en una mujer joven no diabética

Objetivo: Comunicar un caso de cetoacidosis inducida por corticoides y gatifloxacina y discutir los mecanismos de esta inusual y seria complicación. Caso clínico: Mujer de 32 años, ingresa por neumonía adquirida en la comunidad de 5 días de evolución. Antecedentes: AR probable diagnosticada 4 meses antes tratada con metotrexate y corticoides intermitente. Examen físico: regular estado general, IMC 21, Tº 38ºC, FR 32/min, derrame pleural derecho, FC 96/min, PA 110/70, artralgias sin artritis. Exámenes complementarios: Hto 23%, GB 16300/mm3, VSG 96mm/1ºh, glucemia 0.90mg/dl, función hepática y amilasa normales, uremia 1.19g/l, creatinina 19mg/l. Hemocultivos (2) y esputo positivos para Neumococo penicilina-sensible. La neumonía responde a gatifloxacina. Deteriora la función renal hasta la anuria con acidosis metabólica. Se interpreta como glomerulonefritis lúpica rápidamente progresiva por proteinuria de 2g/24hs, FR (+) 1/1280, FAN (+) 1/320 homogéneo, Anti ADN (+) , complemento bajo: C3 29.4mg/dl y C4 10mg/dl, Ac anti Ro, La, Scl70, RNP y anticardiolipinas positivos. Se indica metilprednisolona EV (3 bolos 1g), complicándose con hiperglucemias de >6 g/l y cetoacidosis con cetonuria (+); Ac anti ICA y antiGAD negativos con HbA1C 5.2%. Es tratada en UTI (insulina y hemodiálisis). La paciente mejora, se desciende la dosis de corticoides, con normalización de la glucemia sin tratamiento hipoglucemiante. Comentarios 1) La presencia de HbA1C nomal, Ac anti ICA y GAD negativos permite descartar con razonable grado de certeza una diabetes tipo1 asociada al lupus. 2) El desarrollo de la cetoacidosis durante el tratamiento con corticoides y gatifloxacina y su resolución posterior avalan el rol etiológico de los mismos. 3) La cetoacidosis puede explicarse por estimulación de la gluconeogénesis y la insulinoresistencia a nivel de receptor y post-receptor generada por los fármacos potenciado por el estado inflamatorio relacionado con el lupus y la sepsis.

Cetoacidosis normoglucémica en una embarazada

La cetoacidosis normoglucémica se define como un cuadro de cetoacidosis diabética, con niveles de bicarbonato menores a 10 mEq/l, cetonemia o cetonuria y niveles de glucosa inferiores a 200 mg/dl. Representa hasta el 30% de las formas de presentación de cetoacidosis diabética. Se comunica un caso de cetoacidosis normoglucémica en una embarazada con el objeto de destacar esta inusual asociación y forma de presentación y realizar comentarios respecto a su fisiopatología y tratamiento. Los factores que predisponen a esta condición son los vómitos excesivos, la persistencia del uso de la insulina, una inadecuada ingesta de carbohidratos y el embarazo en algunos casos. El déficit relativo de insulina, la glucogenólisis acelerada con depleción de los depósitos hepáticos de glucosa y la acción del glucagón y hormonas contrainsulares sobre la gluconeogénesis y la lipólisis, son los principales mecanismos responsables del desarrollo de la cetoacidosis euglucémica. El manejo de esta condición es diferente por la necesidad de aporte de volumen con dextrosa al 5-10% en agua y elevado requerimiento de insulina para corregir la cetogénesis y la acidosis. Es importante conocer que aún con glucemias normales puede existir cetoacidosis en pacientes con las condiciones predisponentes mencionadas.

11β-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11β-hydroxysteroid dehydrogenases (11β-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11β-HSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11β-HSD-1) may function as an 11β-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo, mice were produced with targeted disruption of the 11β-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo. Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11β-HSD-1 −/− mice were found to resist hyperglycamia provoked by obesity or stress. Attenuation of hepatic 11β-HSD-1 may provide a novel approach to the regulation of gluconeogenesis.

Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Correction of diabetic alterations by glucokinase.

Hyperglycemia is a common feature of diabetes mellitus. It results from a decrease in glucose utilization by the liver and peripheral tissues and an increase in hepatic glucose production. Glucose phosphorylation by glucokinase is an initial event in glucose metabolism by the liver. However, glucokinase gene expression is very low in diabetic animals. Transgenic mice expressing the P-enolpyruvate carboxykinase/glucokinase chimeric gene were generated to study whether the return of the expression of glucokinase in the liver of diabetic mice might prevent metabolic alterations. In contrast to nontransgenic mice treated with streptozotocin, mice with the transgene previously treated with streptozotocin showed high levels of both glucokinase mRNA and its enzyme activity in the liver, which were associated with an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in gluconeogenesis and ketogenesis in the liver and the production of glucose and ketone body by hepatocytes in primary culture were observed in streptozotocin-treated transgenic mice. Thus, glycolysis was induced while gluconeogenesis and ketogenesis were blocked in the liver of diabetic mice expressing glucokinase. This was associated with normalization of blood glucose, ketone bodies, triglycerides, and free fatty acids even in the absence of insulin. These results suggest that the expression of glucokinase during diabetes might be a new approach to the normalization of hyperglycemia.

Prevention of diabetic alterations in transgenic mice overexpressing Myc in the liver.

Recent studies have demonstrated that the overexpression of the c-myc gene in the liver of transgenic mice leads to an increase in both utilization and accumulation of glucose in the liver, suggesting that c-Myc transcription factor is involved in the control of liver carbohydrate metabolism in vivo. To determine whether the increase in c-Myc might control glucose homeostasis, an intraperitoneal glucose tolerance test was performed. Transgenic mice showed lower levels of blood glucose than control animals, indicating that the overexpression of c-Myc led to an increase of blood glucose disposal by the liver. Thus, the increase in c-Myc might counteract diabetic hyperglycemia. In contrast to control mice, transgenic mice treated with streptozotocin showed normalization of concentrations of blood glucose, ketone bodies, triacylglycerols and free fatty acids in the absence of insulin. These findings resulted from the normalization of liver metabolism in these animals. While low glucokinase activity was detected in the liver of diabetic control mice, high levels of both glucokinase mRNA and enzyme activity were noted in the liver of streptozotocin-treated transgenic mice, which led to an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in the control of gluconeogenesis and ketogenesis and the production of glucose and ketone bodies was observed in streptozotocin-treated transgenic mice. Thus, these results suggested that c-Myc counteracted diabetic alterations through its ability to induce hepatic glucose uptake and utilization and to block the activation of gluconeogenesis and ketogenesis.

Definition of a metal-dependent/Li(+)-inhibited phosphomonoesterase protein family based upon a conserved three-dimensional core structure.

Inositol polyphosphate 1-phosphatase, inositol monophosphate phosphatase, and fructose 1,6-bisphosphatase share a sequence motif, Asp-Pro-(Ile or Leu)-Asp-(Gly or Ser)-(Thr or Ser), that has been shown by crystallographic and mutagenesis studies to bind metal ions and participate in catalysis. We compared the six alpha-carbon coordinates of this motif from the crystal structures of these three phosphatases and found that they are superimposable with rms deviations ranging from 0.27 to 0.60 A. Remarkably, when these proteins were aligned by this motif a common core structure emerged, defined by five alpha-helices and 11 beta-strands comprising 155 residues having rms deviations ranging from 1.48 to 2.66 A. We used the superimposed structures to align the sequences within the common core, and a distant relationship was observed suggesting a common ancestor. The common core was used to align the sequences of several other proteins that share significant similarity to inositol monophosphate phosphatase, including proteins encoded by fungal qa-X and qutG, bacterial suhB and cysQ (identical to amtA), and yeast met22 (identical to hal2). Evolutionary comparison of the core sequences indicate that five distinct branches exist within this family. These proteins share metal-dependent/Li(+)-sensitive phosphomonoesterase activity, and each predicted tree branch exhibits unique substrate specificity. Thus, these proteins define an ancient structurally conserved family involved in diverse metabolic pathways including inositol signaling, gluconeogenesis, sulfate assimilation, and possibly quinone metabolism. Furthermore, we suggest that this protein family identifies candidate enzymes to account for both the therapeutic and toxic actions of Li+ as it is used in patients treated for manic depressive disease.

Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoeno/pyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth. © 2007 by The National Academy of Sciences of the USA.

Metformin

Metformin is the only biguanide antihyperglycemic agent used in the treatment of type 2 (non-insulin dependent) diabetes mellitus. It counters insulin resistance partly by increased insulin action (so-called insulin sensitizing effects) and partly via actions that are not directly insulin dependent. Metformin reduces hepatic glucose output by suppression of gluconeogenesis and glycogenolysis. In skeletal muscle, metformin increases insulin-mediated glucose uptake and glycogen storage. Other actions of metformin that contribute to its blood glucose-lowering effect are reduced fatty acid oxidation and increased glucose turnover, the latter occurring particularly in the splanchnic bed .... © 2007 Copyright © 2007 Elsevier Inc.

Functional genomic and molecular analyses of transcripts related to glycerol synthesis and trafficking in rainbow smelt (Osmerus mordax) using cold temperature-induced models of glycerol production

The rainbow smelt (Osmerus mordax) is an anadromous teleost that produces type II antifreeze protein (AFP) and accumulates modest urea and high glycerol levels in plasma and tissues as adaptive cryoprotectant mechanisms in sub-zero temperatures. It is known that glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature; however, the precise mechanisms of glycerol synthesis and trafficking in smelt remain to be elucidated. The objective of this thesis was to provide further insight using functional genomic techniques [e.g. suppression subtractive hybridization (SSH) cDNA library construction, microarray analyses] and molecular analyses [e.g. cloning, quantitative reverse transcription - polymerase chain reaction (QPCR)]. Novel molecular mechanisms related to glyceroneogenesis were deciphered by comparing the transcript expression profiles of glycerol (cold temperature) and non-glycerol (warm temperature) accumulating hepatocytes (Chapter 2) and livers from intact smelt (Chapter 3). Briefly, glycerol synthesis can be initiated from both amino acids and carbohydrate; however carbohydrate appears to be the preferred source when it is readily available. In glycerol accumulating hepatocytes, levels of the hepatic glucose transporter (GLUT2) plummeted and transcript levels of a suite of genes (PEPCK, MDH2, AAT2, GDH and AQP9) associated with the mobilization of amino acids to fuel glycerol synthesis were all transiently higher. In contrast, in glycerol accumulating livers from intact smelt, glycerol synthesis was primarily fuelled by glycogen degradation with higher PGM and PFK (glycolysis) transcript levels. Whether initiated from amino acids or carbohydrate, there were common metabolic underpinnings. Increased PDK2 (an inhibitor of PDH) transcript levels would direct pyruvate derived from amino acids and / or DHAP derived from G6P to glycerol as opposed to oxidation via the citric acid cycle. Robust LIPL (triglyceride catabolism) transcript levels would provide free fatty acids that could be oxidized to fuel ATP synthesis. Increased cGPDH (glyceroneogenesis) transcript levels were not required for increased glycerol production, suggesting that regulation is more likely by post-translational modification. Finally, levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were transiently higher. These comparisons also led to the novel discoveries that increased G6Pase (glucose synthesis) and increased GS (glutamine synthesis) transcript levels were part of the low temperature response in smelt. Glucose may provide increased colligative protection against freezing; whereas glutamine could serve to store nitrogen released from amino acid catabolism in a non-toxic form and / or be used to synthesize urea via purine synthesis-uricolysis. Novel key aspects of cryoprotectant osmolyte (glycerol and urea) trafficking were elucidated by cloning and characterizing three aquaglyceroporin (GLP)-encoding genes from smelt at the gene and cDNA levels in Chapter 4. GLPs are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. The highlight was the discovery that AQP10ba transcript levels always increase in posterior kidney only at low temperature. This AQP10b gene paralogue may have evolved to aid in the reabsorption of urea from the proximal tubule. This research has contributed significantly to a general understanding of the cold adaptation response in smelt, and more specifically to the development of a working scenario for the mechanisms involved in glycerol synthesis and trafficking in this species.

The dual effect of normobaric hypoxia on heart rate variability and substrate partitioning following interval cycling

Recent studies have shown the importance of the beat-by-beat changes in heart rate influenced by the autonomic nervous system (ANS), or heart rate variability (HRV). The purpose of this study was to examine the lasting effects of hypoxic exercise on HRV, and its influences on substrate usage. Results from this study could lead an increased understanding on this topic. Eight active healthy males (age: 31±11 years; height: 180±7 cm; weight: 83±8 kg; VO₂max (maximal oxygen consumption): 4.4±0.6 L•min⁻¹) underwent normoxic and hypoxic (FᵢO₂= 0.15) conditions during high-intensity interval (HIIT) cycling (70%-high interval, 35%-rest interval). Cycling intensity was determined by a peak power output cycling test. Each experimental session consisted of a basal metabolic rate determination, up to 45-minutes of HIIT cycling, and three 30-minute post-exercise metabolic rate measurements (spanning 3 hours and 15 minutes after exercise). During exercise, RPE was higher (p<0.01) and LAC (lactate) increased (p=0.001) at each point of time in hypoxia, with no change in normoxia. After hypoxic exercise, the SNS/PNS ratio (overall ANS activity) was significantly higher (p<0.01) and significantly decreased through time in both conditions (p<0.01). In addition, a significant interaction between time and conditions (p<0.02) showed a decrease in LAC concentration through time post-hypoxic exercise. The findings showed that a single bout of hypoxic exercise alters ANS activity post-exercise along with shifting substrate partitioning from glycolytic to lipolytic energy production. The significant decrease in LAC concentration post-hypoxic exercise supports the notion that hypoxic HIIT induces a greater muscle glycogen depletion leading to increased fat oxidation to sustain glycogenesis and gluconeogenesis to maintain blood glucose level during recovery.

Comprehensive Transcriptome Analysis of Sex-Biased Expressed Genes Reveals Discrete Biological and Physiological Features of Male and Female Schistosoma japonicum

Schistosomiasis is a chronic and debilitating disease caused by blood flukes (digenetic trematodes) of the genus Schistosoma. Schistosomes are sexually dimorphic and exhibit dramatic morphological changes during a complex lifecycle which requires subtle gene regulatory mechanisms to fulfil these complex biological processes. In the current study, a 41,982 features custom DNA microarray, which represents the most comprehensive probe coverage for any schistosome transcriptome study, was designed based on public domain and local databases to explore differential gene expression in S. japonicum. We found that approximately 1/10 of the total annotated genes in the S. japonicum genome are differentially expressed between adult males and females. In general, genes associated with the cytoskeleton, and motor and neuronal activities were readily expressed in male adult worms, whereas genes involved in amino acid metabolism, nucleotide biosynthesis, gluconeogenesis, glycosylation, cell cycle processes, DNA synthesis and genome fidelity and stability were enriched in females. Further, miRNAs target sites within these gene sets were predicted, which provides a scenario whereby the miRNAs potentially regulate these sex-biased expressed genes. The study significantly expands the expressional and regulatory characteristics of gender-biased expressed genes in schistosomes with high accuracy. The data provide a better appreciation of the biological and physiological features of male and female schistosome parasites, which may lead to novel vaccine targets and the development of new therapeutic interventions.

Energy and Antioxidant Responses of Pacific Oyster Exposed to Trace Levels of Pesticides

Here, we assess the physiological effects induced by environmental concentrations of pesticides in Pacific oyster Crassostrea gigas. Oysters were exposed for 14 d to trace levels of metconazole (0.2 and 2 mu g/L), isoproturon (0.1 and 1 mu g/L), or both in a mixture (0.2 and 0.1 mu g/L, respectively). Exposure to trace levels of pesticides had no effect on the filtration rate, growth, and energy reserves of oysters. However, oysters exposed to metconazole and isoproturon showed an overactivation of the sensing-kinase AMP-activated protein kinase alpha (AMPK alpha), a key enzyme involved in energy metabolism and more particularly glycolysis. In the meantime, these exposed oysters showed a decrease in hexokinase and pyruvate kinase activities, whereas 2-DE proteomic revealed that fructose-1,6-bisphosphatase (F-1,6-BP), a key enzyme of gluconeogenesis, was upregulated. Activities of antioxidant enzymes were higher in oysters exposed to the highest pesticide concentrations. Both pesticides enhanced the superoxide dismutase activity of oysters. Isoproturon enhanced catalase activity, and metconazole enhanced peroxiredoxin activity. Overall, our results show that environmental concentrations of metconazole or isoproturon induced subtle changes in the energy and antioxidant metabolisms of oysters.

Surgical infection and malnutrition

Background: Malnutrition in surgical patients is associated with delayed recovery, higher rates of morbidity and mortality, prolonged hospital stay, increased healthcare costs and a higher early re-admission rate. Methods: Data synthesis after review of pertinent literature. Results: The aetiology of malnutrition is multifactorial. In cancer patients, there is an abnormal peripheral glucose disposal, gluconeogenesis, and whole-body glucose turnover. Malnourished cancer patients undergoing major operations are at significant risk from perioperative complications such as infectious complications. Surgical aggression generates an inflammatory response which worsens intermediary metabolism. Conclusions: Nutritional evaluation and nutritional support must be performed in all surgical patients, in order to minimize infectious complications. Enteral nutrition early in the postoperative period is effective and well tolerated reducing infectious complications, improving wound healing and reducing length of hospital stay. Pharmaconutrition is indicated in those patients, who benefit from enteral administration of arginine, omega 3 and RNA, as well as parenteral glutamine supplementation. When proximal sutures are used, tubes allowing early jejunal feeding should be used.