PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1del/del mice, unmasking role in non-gluconeogenic tissues

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism (AU)

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PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1 del/del mice, unmasking role in non-gluconeogenic tissues.

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.

PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C mediated gluconeogenesis.

BACKGROUND & AIMS: Hepatic gluconeogenesis helps maintain systemic energy homeostasis by compensating for discontinuities in nutrient supply. Liver-specific deletion of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) abolishes gluconeogenesis from mitochondrial substrates, deregulates lipid metabolism and affects TCA cycle. While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). Despite clear relevance to human physiology, the role of PEPCK-M and its gluconeogenic potential remain unknown. Here, we test the significance of PEPCK-M in gluconeogenesis and TCA cycle function in liver-specific PEPCK-C knockout and WT mice. METHODS: The effects of the overexpression of PEPCK-M were examined by a combination of tracer studies and molecular biology techniques. Partial PEPCK-C re-expression was used as a positive control. Metabolic fluxes were evaluated in isolated livers by NMR using (2)H and (13)C tracers. Gluconeogenic potential, together with metabolic profiling, was investigated in vivo and in primary hepatocytes. RESULTS: PEPCK-M expression partially rescued defects in lipid metabolism, gluconeogenesis and TCA cycle function impaired by PEPCK-C deletion, while ∼10% re-expression of PEPCK-C normalized most parameters. When PEPCK-M was expressed in the presence of PEPCK-C, the mitochondrial isozyme amplified total gluconeogenic capacity, suggesting autonomous regulation of oxaloacetate to phosphoenolpyruvate fluxes by the individual isoforms. CONCLUSIONS: We conclude that PEPCK-M has gluconeogenic potential per se, and cooperates with PEPCK-C to adjust gluconeogenic/TCA flux to changes in substrate or energy availability, hinting at a role in the regulation of glucose and lipid metabolism in the human liver.

Nitric oxide as a mediator of fructose 1,6-bisphosphate protection in galactosamine-induced hepatotoxicity in rats.

Fructose 1,6-bisphosphate (F1,6BP) has been widely used as a therapeutic agent for different harmful conditions in a variety of tissues. The hypothesis of the present work was that the increase in nitric oxide production and the prevention of oxidative stress induced by exogenous F1,6BP mediate its protective effect against the hepatotoxic action of GalN. Experimental groups used were sham, F1,6BP (2g/kg bw i.p.), GalN (0.4g/kg bw i.p), l-NAME (10mg/kg bw i.v.), F1,6BP+GalN, l-NAME+GalN and l-NAME+F1,6BP+GalN. Animals were killed after 24h of bolus administration. F1,6BP induced an increase in NO and the redox ratio (GSH/GSSG) in liver. Western blot assays pointed to overexpression of liver eNOS in F1,6BP-treated rats. The hepatic injury induced by GalN increased transaminases in plasma and decreased the reduced/oxidized glutathione ratio in liver. The concomitant administration of F1,6BP reversed this damage, while the addition of l-NAME worsened the liver injury. We provided evidence that this F1,6BP-induced protection may be related to the increase in NO production through the positive modulation of eNOS, and the increase in intracellular reduced glutathione, thus providing a higher reducing capacity.

Functionally enhanced siRNA targeting TNFα attenuates DSS-induced colitis and TLR-mediated immunostimulation in mice.

Tumor necrosis factor (TNFα) is a proinflammatory cytokine involved in the pathogenesis of inflammatory bowel disease (IBD). Although TNFα has been extensively targeted using systemic drugs, the use of antisense and small interfering RNA (siRNA) to drive down its expression at the site of inflammation should provide important advantages. In this study, native and chemically modified siRNA against TNFα was developed and characterized using a murine model of IBD. siRNA with 2'-O-methyl and propanediol modifications (siTNF-OMe-P) were resistant to nuclease degradation and provided better silencing efficacy in vitro as compared to unmodified siRNA. Every modification reduced nonspecific Toll-like receptor (TLR)-mediated immunomodulation in human peripheral blood mononuclear cells (PBMC) cells. Intrarectal administration of siTNF-OMe-P significantly ameliorated the clinical endpoints and histopathological severity in 5% dextran sulphate sodium (DSS)-treated mice as compared to unmodified and other chemically modified siRNAs. Differential gene expression assessed in siTNF-OMe-P-treated animals correlated with improved colon integrity and reduced TLR activation as compared to all treatment groups. All in all, this study demonstrates that propanediol and 2'-O-methyl modifications have profound functional consequences for siRNA efficacy in vivo. Consequently, this strategy has potential implications for therapeutic intervention in IBD and other diseases.

Liver Glucokinase(A456V) Induces Potent Hypoglycemia without Dyslipidemia through a Paradoxical Induction of the Catalytic Subunit of Glucose-6-Phosphatase.

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GK(A456V), described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulation in vivo. GK(A456V) was overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affected in vivo, demonstrating impaired GKRP regulation. GK(A456V) overexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GK(A456V)-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy.

Fructose 1,6 biphosphate administration to rats prevents metabolic acidosis and oxidative stress induced by deep hypothermia and rewarming.

Fructose 1,6 biphosphate (F1,6BP) exerts a protective effect in several in vitro models of induced injury and in isolated organs; however, few studies have been performed using in vivo hypothermia. Here we studied the effects of deep hypothermia (21ºC) and rewarming in anaesthetised rats after F1,6BP administration (2 g/kg body weight). Acid-base and oxidative stress parameters (plasma malondialdehyde and glutathione, and erythrocyte antioxidant enzymes) were evaluated. Erythrocyte and leukocyte numbers in blood and plasma nitric oxide were also measured 3 h after F1,6BP administration in normothermia animals. In the absence of F1,6BP metabolic acidosis developed after rewarming. Oxidative stress was also evident after rewarming, as shown by a decrease in thiol groups and in erythrocyte superoxide dismutase, catalase and GSH-peroxidase, which corresponded to an increase in AST in rewarmed animals. These effects were reverted in rats treated with F1,6BP. Blood samples of F1,6BP-treated animals showed a significant increase in plasma nitric oxide 3 h after administration, coinciding with a significant rise in leukocyte number. F1,6BP protection may be due to the decrease in oxidative stress and to the preservation of the antioxidant pool. In addition, we propose that the reduction in extracellular acidosis may be due to improved tissue perfusion during rewarming and that nitric oxide may play a central role.

Movement evaluation of overerupted upper molars with absolute anchorage: An in-vitro study

Introduction: The overeruption of upper molars due to the premature loss of antagonist teeth can be treated with the help of miniscrews. The aim of this study was to evaluate the movement of a typodont molar according tothe biomechanical approach used with miniscrews. Study design: The study was conducted with four plastermodels filled with typodont wax. In each model we used one absolute anchorage on the palatal side and another on the buccal side in different positions, thus generating four different biomechanical systems. A force of 150 gwas applied to each side of the resin tooth. Periapical radiographs were taken preintrusion and immediately aftercompletion of the intrusion. Photographs were taken in both the sagittal and occlusal planes every 3 min. Theradiographic films and photographs were measured and compared. Results: A vertical movement of the molar was observed in all the models, with system 4 showing the greatest movement. Rotation in the occlusal plane only occurred in system 2, while in system 1 there was a change in the axial axis of 37 degrees. Conclusions: The anchorage site and the combination of forces applied may determine the resulting tooth movement (AU)

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Movement evaluation of overerupted upper molars with absolute anchorage: an in-vitro study.

INTRODUCTION: The overeruption of upper molars due to the premature loss of antagonist teeth can be treated with the help of miniscrews. The aim of this study was to evaluate the movement of a typodont molar according to the biomechanical approach used with miniscrews. STUDY DESIGN: The study was conducted with four plaster models filled with typodont wax. In each model we used one absolute anchorage on the palatal side and another on the buccal side in different positions, thus generating four different biomechanical systems. A force of 150 g was applied to each side of the resin tooth. Periapical radiographs were taken preintrusion and immediately after completion of the intrusion. Photographs were taken in both the sagittal and occlusal planes every 3 min. The radiographic films and photographs were measured and compared. RESULTS: A vertical movement of the molar was observed in all the models, with system 4 showing the greatest movement. Rotation in the occlusal plane only occurred in system 2, while in system 1 there was a change in the axial axis of 37 degrees. CONCLUSIONS: The anchorage site and the combination of forces applied may determine the resulting tooth movement.

Nitric oxide metabolite production during exercise in chronic fatigue syndrome: a case-control study.

BACKGROUND: Chronic fatigue syndrome (CFS) is a disabling illness of unknown etiology that is characterized by fatigue associated with a reduced ability to work, lasting for more than 6 months, and accompanied by a specific set of symptoms. The diagnosis remains difficult because of the absence of laboratory tests and is, therefore, made largely on the basis of the symptoms reported by the patient. The aim of this study was to analyze differences in blood nitrate levels in CFS patients and a matched control group after a physical exercise test. METHODS: Forty-four consecutive female patients with CFS and 25 healthy women performed an exercise test using a cycle ergometer with monitoring of cardiopulmonary response. Blood samples were obtained for biochemical analyses of glucose, lactate, and nitrates at the beginning (under resting conditions) and after the maximal and supramaximal tests. RESULTS: Plasma nitrates differed between the groups, with higher values in the CFS group (F = 6.93, p = 0.003). Nitrate concentration increased in relation to workload and reached higher values in the CFS group, the maximum difference with respect to the control group being 295% (t = 4.88, p < 0.001). CONCLUSIONS: The main result of the present study is that nitric oxide (NO) metabolites (nitrates) showed a much higher increase after a maximal physical test in CFS patients than in a group of matched subjects. This combination (exercise plus NO response evaluation) may be useful in the assessment of CFS.

Fructose 1,6-bisphosphate reduced TNF-alpha-induced apoptosis in galactosamine sensitized rat hepatocytes through activation of nitric oxide and cGMP production.

Fructose 1,6-P2 (F1,6BP) protects rat liver against experimental hepatitis induced by galactosamine (GalN) by means of two parallel effects: prevention of inflammation, and reduction of hepatocyte sensitization to tumour necrosis factor-alpha (TNF-alpha). In a previous paper we reported the underlying mechanism involved in the prevention of inflammation. In the present study, we examined the intracellular mechanisms involved in the F1,6BP inhibition of the apoptosis induced by TNF-alpha in parenchyma cells of GalN-sensitized rat liver. We hypothesized that the increased nitric oxide (NO) production in livers of F1,6BP-treated rats mediates the antiapoptotic effect. This hypothesis was evaluated in cultured primary rat hepatocytes challenged by GalN plus tumour necrosis factor-alpha (GalN+TNF-alpha), to reproduce in vitro the injury associated with experimental hepatitis. Our results show a reduction in apoptosis concomitant with an increase in NO production and with a reduction in oxidative stress. In such conditions, guanylyl cyclase is activated and the increase in cGMP reduces the TNF-alpha-induced apoptosis in hepatocytes. These results provide new insights in the protective mechanism activated by F1,6BP and confirm its interest as a hepatoprotective agent.

Beneficial effects of fructose 1,6-biphosphate on hypothermia-induced reactive oxygen species injury in rats.

The release of reactive oxygen species has been described in hypothermic cells and tissues. Fructose 1,6-biphosphate (F1,6-BP) protects tissue stored at cold temperatures. We study the effect of F1,6-BP in vivo administration on anaesthetized rats exposed to cold stress (4 degrees C chamber for 30 min) and rewarming, to see if it alters cold-induced oxidative injury. Body temperatures show that the animals reached moderate hypothermia (26.80+/-0.62 degrees C) after 30 min of cold exposition. A decrease in mean arterial pressure was found. One group of animals was then rewarmed. Both hypothermia and rewarming increased the production of thiobarbituric acid-reactive substances, an index of lipid peroxidation, and reduced the antioxidant levels of plasmatic sulfhydryl groups, as well as decreasing the enzymatic activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase and GSH peroxidase in erythrocytes. Administration of F1,6-BP increased sulfhydryl groups and limited lipid peroxidation in plasma. It furthermore enhanced Cu,Zn-SOD and GSH peroxidase antioxidant activity in erythrocytes and preserved mean arterial pressure. Therefore, F1,6-BP has therapeutic potential based on its ability to reduce free-radical injury resulting from acute cold exposure and rewarming in vivo.

Pck1 gene silencing in the liver improves glycemia control, insulin sensitivity, and dyslipidemia in db/db mice.

OBJECTIVE: Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by Pck1) catalyzes the first committed step in gluconeogenesis. Extensive evidence demonstrates a direct correlation between PEPCK-C activity and glycemia control. Therefore, we aimed to evaluate the metabolic impact and their underlying mechanisms of knocking down hepatic PEPCK-C in a type 2 diabetic model. RESEARCH DESIGN AND METHODS: PEPCK-C gene targeting was achieved using adenovirus-transduced RNAi. The study assessed several clinical symptoms of diabetes and insulin signaling in peripheral tissues, in addition to changes in gene expression, protein, and metabolites in the liver. Liver bioenergetics was also evaluated. RESULTS: Treatment resulted in reduced PEPCK-C mRNA and protein. After treatment, improved glycemia and insulinemia, lower triglyceride, and higher total and HDL cholesterol were measured. Unsterified fatty acid accumulation was observed in the liver, in the absence of de novo lipogenesis. Despite hepatic lipidosis, treatment resulted in improved insulin signaling in the liver, muscle, and adipose tissue. O(2) consumption measurements in isolated hepatocytes demonstrated unaltered mitochondrial function and a consequent increased cellular energy charge. Key regulatory factors (FOXO1, hepatocyte nuclear factor-4alpha, and peroxisome proliferator-activated receptor-gamma coactivator [PGC]-1alpha) and enzymes (G6Pase) implicated in gluconeogenesis were downregulated after treatment. Finally, the levels of Sirt1, a redox-state sensor that modulates gluconeogenesis through PGC-1alpha, were diminished. CONCLUSIONS: Our observations indicate that silencing PEPCK-C has direct impact on glycemia control and energy metabolism and provides new insights into the potential significance of the enzyme as a therapeutic target for the treatment of diabetes.

Fructose 1,6-bisphosphate prevented endotoxemia, macrophage activation, and liver injury induced by D-galactosamine in rats.

OBJECTIVE: Fructose 1,6-bisphosphate (F1,6BP) protects organs against a wide range of challenges involving inflammation. We hypothesized that the primary action of F1,6BP is to prevent macrophage activation and cytokine release. Our aim was to determine the tissue and cellular targets for this bisphosphorylated sugar and to provide new insights into its mechanisms of action. DESIGN: Prospective, controlled laboratory study. SETTING: Animal resource facilities and research laboratory. SUBJECTS: Male Sprague-Dawley rats (200-250 g body weight). INTERVENTIONS: The protective action of F1,6BP was analyzed in galactosamine (GalN)-induced hepatitis in rats. The in vivo effects of F1,6BP were evaluated by changes in transaminase activities, blood endotoxins, and tumor necrosis factor (TNF)-alpha production in GalN-challenged rats. The targets of F1,6BP to reduce macrophage response to lipopolysaccharide (LPS) were determined by correlation between changes in TNF-alpha production and K+ fluxes through cell membrane in primary cultures of Kupffer cells. MEASUREMENTS AND MAIN RESULTS: The in vivo results indicate that F1,6BP treatment prevented GalN-induced injury in liver parenchymal cells. This protection was mainly associated with a reduction of the inflammatory response. F1,6BP prevention of GalN-induced endotoxemia correlated with preclusion of mast cell degranulation and histamine release that preceded the increased plasma endotoxins and liver production of TNF-alpha. In addition, F1,6BP treatment decreased sensitivity to LPS, which reduced the GalN-induced increase in TNF-alpha. The in vitro results show that F1,6BP inhibited Kupffer cell response and reduced TNF-alpha production by preventing LPS-induced K+ channel activation. CONCLUSIONS: The role of exogenous F1,6BP as a K+ channel modulator underlies its antihistaminic and anti-inflammatory action and increases its interest as a protective compound.

Overcoming diabetes-induced hyperglycemia through inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP) with RNAi.

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) is the rate-controlling enzyme in gluconeogenesis. In diabetic individuals, altered rates of gluconeogenesis are responsible for increased hepatic glucose output and sustained hyperglycemia. Liver-specific inhibition of PEPCK has not been assessed to date as a treatment for diabetes. We have designed a therapeutic, vector-based RNAi approach to induce posttranscriptional gene silencing of hepatic PEPCK using nonviral gene delivery. A transient reduction of PEPCK enzymatic activity (7.6 +/- 0.6 vs 9.7 +/- 1.1 mU/mg, P < 0.05) that correlated with decreased protein content of up to 50% was achieved using this strategy in diabetic mice. PEPCK partial silencing was sufficient to demonstrate lowered blood glucose (218 +/- 26 vs 364 +/- 33 mg/dl, P < 0.001) and improved glucose tolerance together with decreased circulating FFA (0.89 +/- 0.10 vs 1.44 +/- 0.11 mEq/dl, P < 0.001) and TAG (65 +/- 11 vs 102 +/- 16 mg/dl, P < 0.01), in the absence of liver steatosis or lactic acidosis. SREBP1c was down-regulated in PEPCK-silenced animals, suggesting a role for this pathway in the alterations of lipid metabolism. These data reinforce the significance of PEPCK in sustaining diabetes-induced hyperglycemia and validate liver-specific intervention at the level of PEPCK for diabetes gene therapy.

Aspirin inhibits NF-kappaB activation in a glycolysis-depleted lung epithelial cell line.

Inhibition of glycolysis at the phosphofructo-1-kinase step slows cell growth. For this reason, overexpression of fructose-2,6-bisphosphatase is a potential target for antineoplasic treatments. However, therapeutic objectives may be compromised by side effects of glycolysis restriction, including enhanced resistance to oxidants and tumor necrosis factor-alpha (TNF-alpha), as well as increased activity of the nuclear factor kappa B (NF-kappaB). In this study we evaluated aspirin as an adjuvant drug for glycolysis restriction by overexpression of fructose-2,6-bisphosphatase. The effect of aspirin on antioxidant defences and NF-kappaB activity were evaluated both in control cells and in fructose-2,6-bisphosphatase-overexpressing cells. Interestingly, aspirin-induced inhibition of NF-kappaB activity was greater in transfectants with restricted glycolysis than in control cells. Our results indicate that aspirin is a suitable complement to therapy based on glycolysis restriction to overcome resistance associated with increased NF-kappaB activity and oxidative stress.

Gender dimorphism in rat liver mitochondrial oxidative metabolism and biogenesis.

In the present study, we have investigated gender differences in rat liver mitochondrial oxidative metabolism. Total mitochondrial population (M) as well as the heavy (M1), medium (M3), and light (M8) mitochondrial fractions obtained by means of differential centrifugation steps at 1,000, 3,000, and 8,000 g, respectively, were isolated. Electron microscopic analysis was performed and mitochondrial protein content and cardiolipin levels, mitochondrial O(2) flux, ATP synthase activity, mitochondrial membrane potential, and mitochondrial transcription factor A (TFAM) protein levels were measured in each sample. Our results indicate that mitochondria from females have higher protein content and higher cardiolipin levels, greater respiratory and phosphorylative capacities, and more-energized mitochondria in respiratory state 3. Moreover, protein levels of TFAM were four times greater in females than in males. Gender differences in the aforementioned parameters were more patent in the isolated heavy M1 and M3 mitochondrial fractions. The present study demonstrates that gender-related differences in liver mitochondrial function are due mainly to a higher capacity and efficiency of substrate oxidation, likely related to greater mitochondrial machinery in females than in males, which is in accord with greater mitochondrial differentiation in females.

Glutathione content and adaptation to endogenously induced energy depletion in Mv1Lu cells.

Transfection of genes that code for enzymes of energy metabolism provides alternative models to study the adaptive response to energy restriction induced by endogenous changes instead of by unfavorable environmental conditions. Overexpression of the glycolytic enzyme fructose-2,6-bisphosphatase reduced the content of fructose 2,6-bisphosphate, inducing energy limitation in the mink lung epithelial cell line Mv1Lu. This metabolic stress reduced the ATP available in transfected cells by 20%, which downregulated active ion transport and protein turnover. Ion homeostasis and cell function require concomitant reductions in cell membrane ion permeability and protein damage. Our results indicate that glutathione content linked these features of the adaptive response to the endogenously induced metabolic downregulation.

Enhanced antioxidant defenses and resistance to TNF-alpha in a glycolysis-depleted lung epithelial cell line.

Glycolysis-depleted cells, obtained by stable transfection of fructose 2,6-bisphosphatase in mink lung epithelial cells (Mv1Lu), were less sensitive to serum withdrawal- and TNF-alpha-induced apoptosis than cells transfected with the empty vector pcDNA3 (control cells). We compared the differences in the redox status of the two transfectants and the changes produced by TNF-alpha treatment. The activities of the antioxidant enzymes catalase and glutathione peroxidase, as well as the content of reduced glutathione (GSH) and the activity of the nuclear transcription factor kappa B (NF-kappa B), were higher in pFBPase-2 clones than in control cells in all the conditions tested. TNF-alpha challenge sharpened the differences in glutathione peroxidase activity, GSH/GSSG ratios, and NF-kappa B activation between transfectants. These data indicate that glycolysis restriction at the PFK step protects cells against apoptotic stimuli by increasing the GSH content and NF-kappa B activity. This acquired feature may compromise antineoplastic treatments based on glycolytic depletion.