Inhibition of renal gluconeogenesis contributes to hypoglycaemic action of NADPH oxidase inhibitor, apocynin

NADPH oxidase, catalysing superoxide radical (O₂^?) formation, is considered as a main source of reactive oxygen species in kidneys and its increased activity is supposed to be involved in the development of diabetic nephropathy. The aim of this study was to investigate the effect of NADPH oxidase inhibitor, apocynin, on renal gluconeogenesis, which is an important source of endogenous glucose under diabetic conditions.The following observations were made during the experiments performed on isolated renal proximal tubules of control and alloxan diabetic rabbits: (1) apocynin (200 μM) inhibited the rate of glucose synthesis by 45–80%, depending on the substrate applied; (2) the rate of glucose production was also significantly diminished in the presence of TEMPOL (5 mM), a superoxide radical scavenger, suggesting that the decrease in O₂^? formation might be involved in apocynin-evoked gluconeogenesis inhibition; (3) the activities of phosphoenolpyruvate carboxykinase (PEPCK) and/or aldolase were lowered in the presence of either apocynin or TEMPOL, as concluded from the intracellular levels of gluconeogenic intermediates. The data from in vivo experiments indicated that apocynin treatment (2 g/l of drinking water): (1) significantly (by about 30%) attenuated serum glucose concentration in diabetic rabbits and did not affect glycaemia in control animals; (2) normalized diabetes-stimulated rate of glucose synthesis and slightly inhibited gluconeogenesis in control rabbits; (3) normalized diabetes-increased activity of mitochondrial PEPCK and lowered cytosolic PEPCK activity by about 20% below the value for untreated control animals; (4) slightly decreased the activity of mitochondrial PEPCK and did not change the activity of cytosolic one in control rabbits.Thus, it is concluded that: (1) the inhibition of NADPH oxidase might contribute to lowered rate of renal gluconeogenesis, probably due to decreasing PEPCK activity; (2) inhibition of renal gluconeogenesis is involved in apocynin hypoglycaemic action in vivo; (3) apocynin might be beneficial for diabetes treatment.     

Biosynthesis of Ochratoxin A

Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by radiolabeling experiments in which phenylalanine-1-¹⁴C and sodium acetate-2-¹⁴C were supplied to the fungus in sucrose-yeast extract medium. Results showed that phenylalanine was incorporated unaltered into the phenylalanine moiety of ochratoxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via acetate condensation.     

Energy-dependent uptake of ochratoxin A by mitochondria.

The interaction of ochratoxin A, a mycotoxin produced by Aspergillus ochraceus, with isolated rat liver mitochondria and plasma membranes has been studied. Cell membranes bind [¹⁴C]ochratoxin A poorly and do not show saturation in the concentration range examined. The uptake of the toxin by mitochondria is saturable, with an apparent K_m at 0 °C of 30 nmol/mg of protein. Sonication or freeze-thawing reduces the extent of incorporation by 88%. Ochratoxin A uptake is energy dependent, resulting in a depletion of intramitochondrial ATP. Uncouplers such as m-chlorocarbonylcyanide phenylhydrazone or the respiratory inhibitors rotenone and antimycin A inhibit uptake 60–85%, while ATP reverses the antimycin and rotenone inhibition. Phosphate transport is sensitive to inhibition by the toxin, as measured by Ca²⁺ plus P_istimulated respiration and [³²P]P_i incorporation. In turn, phosphate inhibits nearly completely [¹⁴C]ochratoxin A uptake at 22 °C and causes a concomitant mitochondrial swelling yet is not incorporated into the matrix space. Thus, the saturable uptake of ochratoxin A is accompanied by a decrease in the energy state and inhibition of P_i transport, which results in deteriorative changes of the mitochondria, as evidenced by large-amplitude swelling.     

New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.     

Phenylalanine-dependent de novo synthesis of phenylalanine ammonia-lyase from Rhodotorula glutinis

A synthetic medium was developed in which the presence of phenylalanine ammonialyase (PAL) in the yeast Rhodotorula glutinis was dependent on the addition of l-phenylalanine. The appearance of PAL activity occurred during mid- to late log phase regardless of the time of l-phenylalanine introduction into the medium. Maximum levels of PAL activity were followed by a rapid decline in both total and specific activity. These changes were accompanied by comparable fluctuations in PAL antigen levels as measured by rocket immunoelectrophoresis. Proteins of yeast grown in the presences of l-phenylalanine were radiolabeled in vivo with l-[³H]leucine. The labeled protein was immunoprecipitated with anti-PAL serum and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A labeled protein comigrated with subunits of authentic PAL. These data support the hypothesis that de novo synthesis of PAL in R. glutinis is l-phenylalanine dependent.     

Studies of regulatory metabolism in Moniezia expansa: the role of phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase (PEPCK) from M. expansa has been partially purified and its behaviour in a range of different assay conditions has been determined. Different PEPCK's were found in the cytosol and mitochondria. Some kinetic parameters for each are presented. Both enzymes are activated by Mn²⁺; cytosolic PEPCK is also activated by Mg²⁺. The enzymes have pH optima in the range 6·4–7·0. They do not differ with respect to their apparent affinities for inosine and guanosine diphosphates, but the latter allows higher maximal activity. Little activity is observed with adenosine diphosphate. Adenosine and inosine triphosphates exert weak inhibitory effects on the Mn²⁺ activated enzymes; a much strongsr inhibition is exerted on the cytosolic enzyme when activated by Mg²⁺. A number of non-nucleotide compounds were tested for possible inhibitory effects with no success. The forward and back reactions catalyzed by PEPCK proceed at similar rates, suggesting that the enzyme may be readily raversible in vivo.     

NRAGE: A potential rheostat during branching morphogenesis

Branching morphogenesis is a developmental process characteristic of many organ systems. Specifically, during renal branching morphogenesis, its been postulated that the final number of nephrons formed is one key clinical factor in the development of hypertension in adulthood. As it has been established that BMPs regulate, in part, renal activity of p38 MAP kinase (p38^MAPK) and it has demonstrated that the cytoplasmic protein Neurotrophin Receptor MAGE homologue (NRAGE) augments p38^MAPK activation, it was hypothesized that a decrease in the expression of NRAGE during renal branching would result in decreased branching of the UB that correlated with changes in p38^MAPK activation. To verify this, the expression of NRAGE was reduced in ex vivo kidney explants cultures using antisense morpholino. Morpholino treated ex vivo kidney explants expression were severely stunted in branching, a trait that was rescued with the addition of exogenous GDNF. Renal explants also demonstrated a precipitous drop in p38^MAPK activation that too was reversed in the presence of recombinant GDNF. RNA profiling of NRAGE diminished ex vivo kidney explants resulted in altered expression of GDNF, Ret, BMP7 and BMPRIb mRNAs. Our results suggested that in early kidney development NRAGE might have multiple roles during renal branching morphogenesis through association with both the BMP and GDNF signaling pathways.     

Induction of Phosphoenolpyruvate Carboxykinase (PEPCK) during Acute Acidosis and Its Role in Acid Secretion by V-ATPase-Expressing Ionocytes

Vacuolar-Type H⁺-ATPase (V-ATPase) takes the central role in pumping H⁺ through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H⁺ secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H⁺ secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H⁺ via V-ATPase at the cellular level.     

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity

Aims

Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and N^ε-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger.

Methods

Mice were orally administered water or metformin for 15 days (12 or 24 mg kg^− 1 day^− 1), and injected AA (5 mg kg^− 1 day^− 1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed.

Key findings

Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p < 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p < 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity.

Significance

This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Messenger RNA-controlled Increase of Phenylalanine Ammonia-Lyase Activity in Parsley: Light-Independent Induction by Dilution of Cell Suspension Cultures into Water

A specific antiserum, raised against purified phenylalanine ammonialyase from irradiated cell suspension cultures of parsley (Petroselinum hortense Hoffm.), was used to compare the enzyme species induced either by dilution or by irradiation of the cell suspensions, to investigate the effect of dilution on the rate of synthesis of the enzyme protein in vivo, and to analyze the changes in specific activity of polyribosomal mRNA for the enzyme subunits in vitro. The mRNA activity in vitro was measured by translation of the polyribosomal RNA in a rabbit reticulocyte lysate.     

Effect of diphtheria toxin fragment A on energy coupling in mitochondria. Studies on mouse liver mitoplasts

The effect of intact diphtheria toxin and of its fragment A on protein synthesis in mouse liver mitoplasts (digitonin-treated mitochondria) was studied. Fragment A inhibited protein synthesis in intact mitoplasts to the same extent as the uncoupler, carbonylcyanidep-trifluoromethoxyphenylhydrazone, but similar effects were not observed in lyzed mitoplasts. Intact diphtheria toxin was without effect in either case.Fragment A strongly stimulated mitochondrial ATPase activity. At concentrations which efficiently inhibited mitochondrial protein synthesis and stimulated ATPase activity, fragment A had no effect on the intramitochondrial concentration of nicotin-amide adenine dinucleotides. Moreover, it did not catalyze ADP ribosylation of mitochondrial proteins. The results indicate that the effects observed did not involve the NAD⁺-glycohydrolase activity of fragment A.[¹²⁵I]-Labelled fragment A was bound to mitoplasts to about the same extent as the labelled intact diphtheria toxin.The present results suggest that fragment A of diphtheria toxin is capable of inhibiting the energy coupling in mitoplasts, thereby inhibiting protein synthesis. The detailed mechanism of the uncoupling and its possible physiological significance remains to be elucidated     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

Evidence for in vivo compartmentation of phenylalanyl-tRNA ligase in amphibian oocytes

The in vivo activity of phenylalanyl-tRNA ligase of Xenopus laevis oocytes was assayed by measuring the esterification of microinjected yeast tRNA^Phe with [¹⁴C]phenylalanine added to the extracellular medium. The three enzyme substrates, ATP, phenylalanine, and tRNA^Phe, are present in the in vivo assay at saturating concentrations as seen by the fact that microinjection into the cell of additional amounts of these compounds does not increase the quantity of [¹⁴C]Phe-tRNA^Phe formed. The in vivo activity of Phe-tRNA ligase in oocytes at several stages of development is less than 10% of the in vitro activity measured in homogenates of the same cells. The in vivo assay of Phe-tRNA ligase in oocytes that have been microinjected with this enzyme partially purified from X. laevis ovary shows that the enzyme is not inhibited by the cellular conditions. The conclusion drawn from these experiments is that a large fraction of the Phe-tRNA ligase present in oocytes is in a cellular compartment which is not available to the injected tRNA.     

The fate of the larval storage protein calliphorin during adult development of Calliphora vicina

Purified calliphorin labelled with [¹⁴C]phenylalanine by in vivo synthesis was injected into mature Calliphora vicina larvae to examine the distribution of label during metamorphosis and in young adults. About 2.5% of the administered radioactivity was expired during adult development, and 10% was incorporated in the puparium. At the middle of adult development calliphorin was the only detectable radioactive soluble protein in contrast to the situation in the teneral adult where most soluble proteins were labelled. All organs and/or tissues of 4-day flies were radioactive; nearly half the phenylalanine of calliphorin was incorporated into flight muscle, actin and myosin being strongly labelled. Earlier hypotheses that calliphorin is a larval storage protein are now experimentally verified.     

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells has been studied. When these cells are synchronized for initiation by fluoride treatment and then double labeled with [³⁵S]methionine and either tritiated proline, phenylalanine, or valine for short pulses, the percentage of N-terminal methionine incorporated in the nascent peptides compared to total incorporation is significantly higher than that of the tritiated amino acids tested. The data indicate that methionine is the initiator amino acid for the synthesis of poliovirus-specific proteins.