Forkhead Box O6 (FoxO6) Depletion Attenuates Hepatic Gluconeogenesis and Protects against Fat-induced Glucose Disorder in Mice

Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. FoxO6 is a distinct member of the FoxO subfamily. To elucidate the role of FoxO6 in hepatic gluconeogenesis and assess its contribution to the pathogenesis of fasting hyperglycemia in diabetes, we generated FoxO6 knock-out (FoxO6-KO) mice followed by determining the effect of FoxO6 loss-of-function on hepatic gluconeogenesis under physiological and pathological conditions. FoxO6 depletion attenuated hepatic gluconeogenesis and lowered fasting glycemia in FoxO6-KO mice. FoxO6-deficient primary hepatocytes were associated with reduced capacities to produce glucose in response to glucagon. When fed a high fat diet, FoxO6-KO mice exhibited significantly enhanced glucose tolerance and reduced blood glucose levels accompanied by improved insulin sensitivity. These effects correlated with attenuated hepatic gluconeogenesis in FoxO6-KO mice. In contrast, wild-type littermates developed fat-induced glucose intolerance with a concomitant induction of fasting hyperinsulinemia and hyperglycemia. Furthermore, FoxO6-KO mice displayed significantly diminished macrophage infiltration into liver and adipose tissues, correlating with the reduction of macrophage expression of C-C chemokine receptor 2 (CCR2), a factor that is critical for regulating macrophage recruitment in peripheral tissues. Our data indicate that FoxO6 depletion protected against diet-induced glucose intolerance and insulin resistance by attenuating hepatic gluconeogenesis and curbing macrophage infiltration in liver and adipose tissues in mice.     

Biochemical and structural brain alterations in female mice with cerebral pyruvate dehydrogenase deficiency

Pyruvate dehydrogenase complex (PDC) deficiency is an inborn metabolic disorder associated with a variety of neurologic abnormalities. This report describes the development and initial characterization of a novel murine model system in which PDC deficiency has been introduced specifically into the developing nervous system. The absence of liveborn male and a roughly 50% reduction in female offspring following induction of the X-linked mutation indicate that extensive deficiency of PDC in the nervous system leads to pre-natal lethality. Brain tissue from surviving females at post-natal days 15 and 35 was shown to have approximately 75% of wild-type PDC activity, suggesting that a threshold of enzyme activity exists for post-natal survival. Detailed histological analyses of brain tissue revealed structural defects such as disordered neuronal cytoarchitecture and neuropil fibers in grey matter, and reduced size of bundles and disorganization of fibers in white matter. Many of the histologic abnormalities resemble those found in human female patients who carry mutations in the X-linked ortholog. These findings demonstrate a requirement for PDC activity within the nervous system for survival in utero and suggest that impaired pyruvate metabolism in the developing brain can affect neuronal migration, axonal growth and cell-cell interactions.     

Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets

Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.     

Glutamate Dehydrogenase in Cerebellar Mutant Mice: Gene Localization and Enzyme Activity in Different Tissues

Many similarities of both the inheritance pattern and the neuropathology can be observed between olivopontocerebellar atrophies, or so-called multiple system atrophies (MSAs), and murine cerebellar mutations like Purkinje cell degeneration, nervous, staggerer, weaver, and reeler. Our study aimed to test whether the glutamate dehydrogenase (GDH) deficiency observed in some MSA patients could be found also in any of the murine mutants. GDH activity was assayed in several organs of these mutants, and no general deficiency was detected. By contrast, the level was found to be elevated in the cerebellum. The GDH gene was localized on mouse chromosome 14 and does not map close to any known neurological mutation in the mouse. We conclude, for the moment, that none of these cerebellar mutant mice can be considered as an animal model for GDH-deficient MSA.     

Role of the pyruvate metabolic network on carbohydrate metabolism and virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize different carbohydrates and how this impacts survival in the host. Loss of ldh decreased bacterial burden in the nasopharynx and enhanced bacteremia in mice. Loss of spxB, pdhC or pfl2 decreased bacteremia and increased host survival. In glucose or galactose, loss of ldh increased capsule production, whereas loss of spxB and pdhC reduced capsule production. The pfl2 mutant exhibited reduced capsule production only in galactose. In glucose, pyruvate was metabolized primarily by LDH to generate lactate and NAD⁺ and by SpxB and PDHc to generate acetyl-CoA. In galactose, pyruvate metabolism was shunted toward acetyl-CoA production. The majority of acetyl-CoA generated by PFL was used to regenerate NAD⁺ with a subset used in capsule production, while the acetyl-CoA generated by SpxB and PDHc was utilized primarily for capsule biosynthesis. These data suggest that the pneumococcus can alter flux of pyruvate metabolism dependent on the carbohydrate present to succeed in distinct host niches.     

Changes in lactate content in the gills of the mudskippers Periophthalmus chrysospilos and Boleophthalmus boddaerti in response to environmental hypoxia

The gills of Periophthalmus chrysospilos exhibited significantly greater pyruvate kinase and lactate dehydrogenase activities than those of Boleophthalmus boddaerti ( P <0.01). Under the control normoxic condition, the branchial lactate content of the former mudskipper was also significantly greater ( P < 0.01) than that of the latter. After hypoxic exposure, lactate and alanine accumulated in the gills of P. chrysospilos but not B. boddaerti . Succinate and propionate were not detected in the gills of these two mudskippers under the control and hypoxic conditions.     

Effect of temperature upon catalytic properties of lactate dehydrogenase in the lobster

Lactate dehydrogenase (LDH) present in the tail muscle of the lobster (H. vulgaris) exhibits substrate (pyruvate and L-lactate) inhibition which is temperature-dependent. Such inhibitions can be related to the formation of stable LDH-NAD ⁺-pyruvate and LDH-NADH-lactate complexes. The apparent K_m of pyruvate and L-lactate increase when the temperature rises above 12°. These temperature-dependent kinetic properties may play a major role in determining the metabolic fate of pyruvate.     

Advantages and disadvantages of the animal models v. in vitro studies in iron metabolism: a review

Iron deficiency is the most common nutritional deficiency in the world. Special molecules have evolved for iron acquisition, transport and storage in soluble, nontoxic forms. Studies about the effects of iron on health are focused on iron metabolism or nutrition to prevent or treat iron deficiency and anemia. These studies are focused in two main aspects: (1) basic studies to elucidate iron metabolism and (2) nutritional studies to evaluate the efficacy of iron supplementation to prevent or treat iron deficiency and anemia. This paper reviews the advantages and disadvantages of the experimental models commonly used as well as the methods that are more used in studies related to iron. In vitro studies have used different parts of the gut. In vivo studies are done in humans and animals such as mice, rats, pigs and monkeys. Iron metabolism is a complex process that includes interactions at the systemic level. In vitro studies, despite physiological differences to humans, are useful to increase knowledge related to this essential micronutrient. Isotopic techniques are the most recommended in studies related to iron, but their high cost and required logistic, making them difficult to use. The depletion–-repletion of hemoglobin is a method commonly used in animal studies. Three depletion–-repletion techniques are mostly used: hemoglobin regeneration efficiency, relative biological values (RBV) and metabolic balance, which are official methods of the association of official analytical chemists. These techniques are well-validated to be used as studies related to iron and their results can be extrapolated to humans. Knowledge about the main advantages and disadvantages of the in vitro and animal models, and methods used in these studies, could increase confidence of researchers in the experimental results with less costs.     

Gluconeogenesis and ketogenesis in perfused liver of rats submitted to short-term insulin-induced hypoglycaemia

Gluconeogenesis and ketogenesis of in situ rat perfused liver submitted to short-term insulin-induced hypoglycaemia (IIH) were investigated. For this purpose, 24-h fasted rats that received intraperitoneal (ip) regular insulin (1.0 U kg(-1)) or saline were compared. The studies were performed 30 min after insulin (IIH group) or saline (COG group) injection. For gluconeogenesis studies, livers from the IIH and COG groups were perfused with increasing concentrations (from basal blood concentrations until saturating concentration) of glycerol, L-lactate (Lac) or pyruvate (Pyr). Livers of the IIH group showed maintained efficiency to produce glucose from glycerol and higher efficiency to produce glucose from Lac and Pyr. In agreement with these results the oral administration of glycerol (100 mg kg(-1)), Lac (100 mg kg(-1)), Pyr (100 mg kg(-1)) or glycerol (100 mg kg(-1)) + Lac (100 mg kg(-1)) + Pyr (100 mg kg(-1)) promoted glycaemia recovery. It can be inferred that the increased portal availability of Lac, Pyr and glycerol could help glycaemia recovery by a mechanism mediated, partly at least, by a maintained (glycerol) or increased (Lac and Pyr) hepatic efficiency to produce glucose. Moreover, in spite of the fact that insulin inhibits ketogenesis, the capacity of the liver to produce ketone bodies from octanoate during IIH was maintained.     

肝纤维化动物模型探讨

目的 寻找肝纤维化最佳模型.方法 将Wistar大鼠随机分成血清组、四氯化碳皮下注射组、四氯化碳腹腔注射组,每组30只,各组分别给予猪血清腹腔注射、40%四氯化碳皮下和腹腔注射造模(每周2次),观察造模过程中大鼠死亡情况以及4周及6周各组大鼠肝纤维化的程度.结果 3种方法都能成功制备肝纤维化模型.从动物死亡情况来看,四氯化碳腹腔注射组死亡率明显高于前两组;血清组死亡率最低,但与四氯化碳皮下注射组比较无显著差异;从模型形成时间来看,血清组造模时间较长,明显高于其他两组,四氯化碳皮下注射组与四氯化碳腹腔注射组在模型形成时间上无明显差异.结论 四氯化碳皮下注射组制备肝纤维化模型动物死亡率较低,肝纤维化形成时间较短,是一种制作肝纤维化模型较好的方法.     

Early Atherosclerotic Plaques in the Aorta Following Cytomegalovirus Infection of Mice

We show here that BALB/c mice inoculated with murine cytomegalovirus (MCMV) express viral antigens in the endothelial and smooth muscle cells of the aortic wall, and that accumulation of inflammatory cells in the aortic lumen, similar to that seen in early atherosclerotic lesions in humans, colocalizes with the site of virus antigen expression. Immunosuppression of the mice at the time of virus infection increased the expression of viral antigens and the size of early atherosclerotic lesions in the intima. The percentage of the low-density lipoprotein cholesterol (LDL-C), the major lipid contributor to atherosclerotic plaques, was significantly increased in the serum of MCMV-infected mice, whether or not the mice were fed a high cholesterol diet. Human cytomegalovirus (HCMV) significantly increased the esterified cholesterol component of the total cholesterol in a human arterial smooth muscle cell line infected in vitro with HCMV. These results suggest that CMV infection is involved in two of the major mechanisms that lead to development of atherosclerosis, i.e., immune injury and high LDL-C.     

Activity of Pyrimidine Degradation Enzymes in Normal Tissues

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and ß-ureidopropionase (ß-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of ß-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and ß-UP were also present in a variety of other tissues, especially in bronchus.     

Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier

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Genetic Variation in Activity of the Enzymes of Glycolysis and Gluconeogenesis between Inbred Strains of Mice

Variation in the activity of 21 liver and 15 erythrocyte enzymes between seven inbred strains of mice has been studied in a single area of metabolism, glycolysis and gluconeogenesis. Most of the variation between the strains is genetic. From the variation within and between inbred strains heritabilities (H²) were determined. Out of 35, 26 showed significant values above 0.4. A comparison with previously published work suggests that enzyme activities have mainly dominance and interaction components of variance, and this is discussed in relation to the variation in quantitative characters such as growth. In nine of the pairwise comparisons of the strains, the activity of the enzyme varied more than two-fold. In these cases the genetics and biochemistry of the enzyme was studied; F₂ progeny were produced and assessed for segregation, and the heat stability of the enzyme was determined. No unequivocal segregation was observed, although in one case we found a considerable difference in heat stability. The variations found were not considered to be great enough to be useful as models of human inborn errors of metabolism or to study metabolic control. If such variants are to be found, sources of variation other than inbred strains must be used.     

The metabolic acclimation of Arabidopsis thaliana to arsenate is sensitized by the loss of mitochondrial LIPOAMIDE DEHYDROGENASE2, a key enzyme in oxidative metabolism

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild‐type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady‐state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild‐type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis.     

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: Preparation and characterization

The dihydrolipoyl transacetylase core component of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N, N′-p-phenylenedimaleimide.In one approach, the N, N′-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found the N, N′-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues.Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.     

The activity of the pyruvate dehydrogenase complex in heart muscle in the previously obese mouse model

Obese gold thioglucose injected mice were reduced to lean control weight by food restriction. When pair fed with lean controls these animals then gained weight (were metabolically more efficient). Serum glucose was also elevated in this group (14.5±0.4 (14)vs 12.1±0.3 mmol/L, p<0.001). If previously obese animals were weight maintained with lean controls (by mild food restriction), serum glucose remained at control levels. The activity of the pyruvate dehydrogenase complex in heart muscle was decreased in both obese and pair fed previously obese, whilst it was similar to that of lean controls in the weight maintained previously obese and in obese mice actually dieted. In all obese and previously obese animals serum insulin was elevated. In hearts from control animals subjected to mild food restriction the pyruvate dehydrogenase complex was activated (11.53±1.80 (5)vs 3.34±0.62 (9) U/g dry weight), despite a reduced serum insulin level (42±2vs 74±10 U/ml, p<0.01). These diverse changes in the proportion of the pyruvate dehydrogenase complex in the active form and insulin levels argue for a persistent alteration in the sensitivity of the pyruvate dehydrogenase complex to insulin in obesity, as well as indicating that glucose metabolism in obese animals is altered by both body weight and diet amount.To whom correspondence should be addressed.     

Growth and Carbohydrate Metabolism of Sulfobacilli

The moderately thermophilic acidophilic bacteria Sulfobacillus thermosulfidooxidans, strain 1269, S. thermosulfidooxidanssubsp. asporogenes, strain 41, and the thermotolerant strain S. thermosulfidooxidanssubsp. thermotolerans K1 prefer mixotrophic growth conditions (the concomitant presence of ferrous iron, thiosulfate, and organic compounds in the medium). In heterotrophic and autotrophic growth conditions, these sulfobacilli can grow over only a few culture transfers. In cell-free extracts of these sulfobacilli, key enzymes of the Embden–Meyerhof–Parnas, pentose-phosphate, and Entner–Doudoroff pathways were found. The role of a particular pathway depended on the cultivation conditions. All of the enzymes assayed were most active under mixotrophic conditions in the presence of Fe²⁺and glucose, suggesting the operation of all of the three major pathways of carbohydrate metabolism under these conditions. However, the operation of the Entner–Doudoroff pathway in strain 41 was restricted under mixotrophic conditions. After the first culture transfer from mixotrophic to heterotrophic conditions, the utilization of glucose occurred only via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. After the first culture transfer from mixotrophic to autotrophic conditions, the activity of carbohydrate metabolism enzymes decreased in all of the strains studied; in strain K1, only the glycolytic pathway remained operative. The high activity of fructose-bisphosphate aldolase, remaining in strain 41 cells under these conditions, suggests the involvement of this enzyme in the reactions of the Calvin cycle or of gluconeogenesis.     

Regulation of the pentose phosphate pathway at fertilization in sea urchin eggs

Summary

After fertilization of sea urchin eggs, there is a rapid increase in cellular levels of NADPH, a metabolite utilized in a variety of biosynthetic reactions during early development. Recent studies have shown that a dramatic increase in the activity of the pentose phosphate shunt occurs in vivo shortly after fertilization, consistent with the hypothesis mat this metabolic pathway is a major supplier of NADPH in sea urchin zygotes. One mechanism that may account, in part, for this increase in pentose shunt activity is the dissociation of glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of the shunt, from cell structural elements. In vitro, G6PDH is associated with the insoluble matrix obtained from homogenates of unfertilized eggs, and in this state, the enzyme is inhibited. Within minutes of fertilization, G6PDH is released as an active, soluble enzyme. A similar solubilization and activation of G6PDH occurs after fertilization of eggs of other marine invertebrates and in mammalian cells in culture stimulated by growth factors. The occurrence of this phenomenon in such diverse cell types, in response to different stimuli, suggests that the redistribution of G6PDH between insoluble and soluble locations may be involved in the regulation of the pentose phosphate shunt during cell activation in general.     

Enzymatic and Immunologic Identification of Succinic Semialdehyde Dehydrogenase in Rat and Human Neural and Nonneural Tissues

Abstract: We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.