GlcA-mediated glycerol-3-phosphate synthesis contributes to the oxidation resistance of Aspergillus fumigatus via decreasing the cellular ROS.

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.

Signaling of chloroquine-induced stress in the yeast Saccharomyces cerevisiae requires the Hog1 and Slt2 mitogen-activated protein kinase pathways.

Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.

Adipogenic effect of calcium sensing receptor activation.

We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein ß. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.

Ridostin induces transcription of a wide spectrum of interferon genes in human cells.

The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-ß, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-ß, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of ß-actin, glycerophosphate dehydrogenase, and ß2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.

GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling.

Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes.

The transdehydrogenase genes KlNDE1 and KlNDI1 regulate the expression of KlGUT2 in the yeast Kluyveromyces lactis.

KlNDE1 and KlNDI1 code for two inner mitochondrial membrane transdehydrogenases involved in the maintenance of the intracellular NAD(P)H redox balance. The function of these genes during the utilization of fermentative and respiratory carbon sources was studied. During growth in glucose, deletion of KlNDE1 and KlNDI1 led to an altered kinetic of ethanol and glycerol accumulation compared with the wild type; in addition, KlndiDelta was unable to grow in respiratory substrates. Northern analysis and GFP-fusion experiments showed that KlNDE1 and KlNDI1 regulate the expression of KlGUT2, a component of the glycerol-3-phosphate shuttle. Moreover, both genes seem to be involved in the biogenesis of the mitochondrial tubular network.

[The correlation between fish growth and several biochemical characteristics by the example of the steelhead Parasalmo mykiss Walb].

Correlation analysis demonstrated a statistically significant correlation of linear-weight characteristics of the steelhead rainbow trout (cultivated steelhead form) with an RNA/DNA ratio and the expression level of the gene encoding cytochrome c oxidase (CO) in two-year-old individuals (1+) as well as the expression level of the gene encoding the myosin heavy chain (MyHC) and activities of the enzymes CO and lactate dehydrogenase (LDH) in muscles and 1-glycerophosphate dehydrogenase (1-GPDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the liver of two- and three-year-old individuals (1+ and 2+). With age, the correlation of 1-GPDH and G-6-PDH activities in the rainbow trout liver with the fish body weight increased, whereas their correlations with the body length reduced. The age- and sex-related distinctions in the MyHC gene expression and activities of the white muscle enzyme LDH and the liver enzymes 2-GPDH and G-6-PDH were detected in rainbow trout of both age cohorts.

GPD1 Enhances the Anticancer Effects of Metformin by Synergistically Increasing Total Cellular Glycerol-3-Phosphate.

Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here, we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anticancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy. SIGNIFICANCE: GPD1 overexpression enhances the anticancer effect of metformin through synergistic inhibition of mitochondrial function, thereby providing new insight into metformin-mediated cancer therapy.

Carcinogens 3,4-benzpyrene and 3-methylcholanthrene: induction of mitochondrial oxidative enzymes.

Sections of liver from rats injected with 3,4-benzpyrene and 3-methylcholanthrene, when incubated in mediums specific for the histochemical demonstration of mitochondrial oxidative enzymes, show greater activity of several of these enzymes than do sections from control rats. This observation was confirmed by comparison of the staining of mitochondria isolated from the control and from "induced" rats. The fact that an inhibitor of protein synthesis, actinomycin D, effectively diminished the stimulation provided evidence that the stimulation of activity is due to an increase in enzyme synthesis, generally called induction.

Effects of triethylamine on the expression patterns of two G3PDHs and lipid accumulation in Dunaliella tertiolecta.

Glycerol-3-phosphate (G3P) is the important precursors for triacylglycerol synthesis, while glycerol-3-phosphate dehydrogenase (GPDH) determines the formation of G3P. In this study, two GDPH genes, Dtgdp1 and Dtgdp2 were isolated and identified from Dunaliella tertiolecta. The full-length Dtgdp1 and Dtgdp2 CDS were 2016 bp and 2094 bp, which encoded two putative protein sequences of 671 and 697 amino acids with predicted molecular weights of 73.64 kDa and 76.73 kDa, respectively. DtGDP1 and DtGDP2 both had a close relationship with those of algal and higher plants. DtGDP1 shared two conserved superfamily (A1 and A2) and four signature motifs (I-IV), and the DtGDP2 showed six signature domains (from motif I to VI) and DAO_C conserved family. Our previous work showed that the triethylamine intervention could greatly increase the triacylglycerol content (up to 80%) of D. tertiolecta. This study aims to investigate the effect of triethylamine on GPDH expression. Results showed that, when treated by triethylamine at 100 ppm and 150 ppm, the expression levels of Dtgdp1 and Dtgpd2 were increased to 5.121- and 56.964-fold compared with the control, respectively. Triethylamine seemed to enhance lipid metabolic flow by inducing the expressions of Dtgdp1 and Dtgdp2 to increase the lipid content, which provides a new insight into the desired pathway of lipid synthesis in algae through genetic engineering.

Thyroid hormone--sympathetic interaction and adaptive thermogenesis are thyroid hormone receptor isoform--specific.

In newborns and small mammals, cold-induced adaptive (or nonshivering) thermogenesis is produced primarily in brown adipose tissue (BAT). Heat production is stimulated by the sympathetic nervous system, but it has an absolute requirement for thyroid hormone. We used the thyroid hormone receptor-beta--selective (TR-beta--selective) ligand, GC-1, to determine by a pharmacological approach whether adaptive thermogenesis was TR isoform--specific. Hypothyroid mice were treated for 10 days with varying doses of T3 or GC-1. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was restored by either T3 or GC-1 treatment. However, whereas interscapular BAT in T3-treated mice showed a 3.0 degrees C elevation upon infusion of norepinephrine, indicating normal thermogenesis, the temperature did not increase (<0.5 degrees C) in GC-1--treated mice. When exposed to cold (4 degrees C), GC-1--treated mice also failed to maintain core body temperature and had reduced stimulation of BAT UCP1 mRNA, indicating impaired adrenergic responsiveness. Brown adipocytes isolated from hypothyroid mice replaced with T3, but not from those replaced with GC-1, had normal cAMP production in response to adrenergic stimulation in vitro. We conclude that two distinct thyroid-dependent pathways, stimulation of UCP1 and augmentation of adrenergic responsiveness, are mediated by different TR isoforms in the same tissue.

Time-course of hormonal induction of mitochondrial glycerophosphate dehydrogenase biogenesis in rat liver.

Thyroid hormones are important regulators of mitochondrial metabolism. Due to their complex mechanism of action, the timescale of different responses varies from minutes to days. In this work, we studied selective T3 induction of the inner mitochondrial membrane enzyme-glycerophosphate dehydrogenase (mGPDH) in liver of euthyroid rats. We correlated the kinetics of the T3 level in blood, the mRNA level in liver, the activity and amount of mGPDH in liver mitochondria after a single dose of T3. The T3 level reached maximum after 1 h (80 nmol/l) and subsequently rapidly decreased. mGPDH mRNA increased also relatively fast, reaching a maximum after 12 h and fell to the control level after 72 h. An increase of mGPDH activity could be already found after 6 h and reached a maximum after 24 h in accordance with the increase in mGPDH content (2.4-fold vs. 2.7-fold induction). After 72 h, the mGPDH activity showed a significant 30% decrease. When the rats received three subsequent doses of T3, the increase of mGPDH activity was 2-fold higher than after a single T3 dose. The results demonstrate that mGPDH displays rapid induction as well as decay upon disappearance of a hormonal stimulus, indicating a rather short half-life of this inner mitochondrial membrane enzyme.

The reversible expression of an adult isozyme locus, Gdc-1, in tumors of the mouse.

Tumors of the mouse possess 2 isozymic forms of L-glycerol-3-phosphate dehydrogenase (alpha-GPDH) (EC 1.1.1.8) that can be distinguished from each other by their heat inactivation and electrophoretic properties. These isozymes share certain structural features, since dissociation and reassociation of mixtures of the 2 isozymes lead to the generation of a hybrid molecular species. This finding suggests that the structural genes for these isozymes are closely related. A number of spontaneous and transplantable tumors of the mouse have been analyzed in order to assess whether the pattern of embryonic and adult alpha-GPDH isozyme expression is correlated with the degree of tumor differentiation. The results indicate that no correlation between the type of isozyme expressed and the degree of tumor differentiation or growth rate was evident. A striking correlation exists, however, between the physical form of the tumor and isozyme expression; all solid tumors possess, predominantly, the adult isozymic form of L-glycerol-3-phosphate dehydrogenase, whereas all ascites tumors, including embryoid bodies from ovarian and testicular teratomas, possess the embryonic form. A solid tumor, the C1300 neuroblastoma, that initially possessed the adult isozyme, was cultured in vitro; this resulted in the disappearance of the adult isozyme and predominant expression of the embryonic isozyme. Reinjection of cultured neuroblastoma cells into a host mouse produced a solid tumor that possessed the adult isozyme. The exclusive presence of either adult alpha-GPDH in solid tumor growths or embryonic alpha-GPDH in ascites tumor growths after converting from one physical forms of the tumor to the other, strongly supports a genetic regulatory mechanism which depends on the reversible repression and activation of the structural loci for these isozymes.

The induction of mitochondrial L-3-glycerophosphate dehydrogenase by thyroid hormone.

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.

The synergistic interaction of hydrocortisone and dibutyryl cyclic AMP during enzyme induction in hybrids between rat C6 glioma cells and FU5AH hepatoma cells.

The hormone-responsive enzymes tyrosine aminotransferase and glycerol-3-phosphate dehydrogenase were studied with respect to current models of the mechanism of glucocorticoid/cAMP interaction during the induction of enzyme activity in responsive cell hybrids between rat C6 glioma cells and rat FU5AH hepatoma cells. The results of experiments involving protein and mRNA synthesis inhibitors, sequential addition of inducers, and the assay of cyclic-AMP-dependent protein kinase could not be adequately explained by any one model of inducer interaction. Comparison of the hybrid clones revealed the presence of factors that may modify induction but that are not essential for synergistic induction.

Regulation of mitochondrial glycerol-phosphate dehydrogenase by Ca2+ within electropermeabilized insulin-secreting cells (INS-1).

(1) A new insulin-secreting cell line (INS-1; Asfari et al. (1992) Endocrinology 130, 167-178) has been used to study the regulation by Ca2+ of mitochondrial FAD-linked glycerol-phosphate dehydrogenase (FAD-GPDH) in situ. (2) Enzyme activity was examined on-line in electropermeabilized cells by a new, sensitive, assay. This involved the reduction of the artificial electron acceptor, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), monitored by the quenching of the fluorescence of rhodamine-18. Using this approach, similar total levels of FAD-GPDH activity (nmol/min per 10(6) cells) were measured in INS-1 cells (1.35 +/- 0.22) and isolated rat islet cells (1.63 +/- 0.02) (3) Ca2+ ions markedly activated the enzyme, lowering the apparent Km-value for added DL-glycerophosphate from 8.8 +/- 1.4 mM to 1.0 +/- 0.1 mM. Ca2+ had no effect on the apparent Vmax. The enzyme displayed cooperative kinetics with respect to DL-glycerophosphate (Hill coefficient of 2.0 +/- 0.2 and 1.6 +/- 0.2 in the absence and presence respectively of Ca2+). Half-maximal effects of Ca2+ were observed in the range 30-130 nM, depending on the concentration of glycerol phosphate. (4) Enzyme activity was weakly (30%) inhibited by diazoxide, but not by the diabetogenic drug, streptozotocin. (5) The data indicate that INS-1 cells represent an excellent model for studying the rôle of FAD-GPDH in the control of insulin secretion.

Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells.

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.

Genetic regulation of sn-glycerol-3-phosphate dehydrogenase in brown adipose tissue.

The levels of sn-glycerol-3-phosphate dehydrogenase (GPDH) were determined in the brown adipose tissue (BAT) of different inbred strains of mice. The BAT of the BALB/cJ strain contains twice as much enzyme activity per milligram protein as do other strains. The appearance of this difference is developmentally dependent, since it is not detected in BAT until 25-30 days postpartum. Genetic analysis of this strain difference has shown that the mechanism of inheritance involves at least two genes, one of which is linked to the Gdc-1 structural locus on chromosome 15. Determinations of GPDH synthesis by immunoprecipitation of GPDH protein labeled in vivo with [3H]leucine, and of GPDH mRNA by Northern blot analysis, establish that in BALB/cJ mice higher rates of enzyme synthesis are determined by elevated levels of GPDH mRNA. It was also found that cold stress increases GPDH mRNA levels in all the strains examined.

Proliferation and differentiation of cultured MC3T3-E1 osteoblasts on surface-layer modified hydroxyapatite ceramic with acid and heat treatments.

Effects of functionally gradient calcium phosphate consisting of hydroxyapatite (HAP) and alpha-tricalcium phosphate (alpha-TCP) on proliferation and differentiation of osteoblasts were evaluated using MC3T3-E1 cells. There were no significant differences in the proliferation of MC3T3-E1 cells among HAP-alpha-TCP functionally gradient calcium phosphate, pure HAP, and cell culture plastic wells. mRNA expressions of type I collagen, alkaline phosphate, and osteocalcine were evaluated as indexes of initial; mid-stage, and late-stage osteoblastic differentiation. Basically, HAP-alpha-TCP functionally gradient calcium phosphate and pure HAP enhanced the expressions of the three markers when compared with that of cell culture plastic wells. For type I collagen and alkaline phosphate expressions, HAP-alpha-TCP functionally gradient calcium phosphate showed the same expression level as pure HAP. For osteocalcine expression, HAP-alpha-TCP functionally gradient calcium phosphate showed a higher level than pure HAP. We concluded, therefore, HAP-alpha-TCP functionally gradient calcium phosphate has good potential to be a bone filler material with high osteoconductivity.

Alpha-amanitin administration results in a temporary inhibition of hepatic enzyme induction by triiodothyronine: further evidence favoring a long-lived mediator of thyroid hormone action.

Alpha-amanitin was shown to inhibit triiodothyronine (T3)-induced increases in mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytoplasmic malic enzyme activity in the livers of male Sprague-Dawley rats. A 3-fold increase in alpha-GPD observed 24 h after the iv injection of 3 microngT3/100 g BW was completely inhibited by administration of alpha-amanitin at 0 and 8 h. Similarly, alpha-amanitin blocked a two- to four-fold increase in malic enzyme 24 h following iv injection of 3 mg T3/100 g BW into euthyroid rats. After the initial inhibition of enzyme induction by alpha-amanitin was dissipated, however, a delayed but striking increase in enzyme activity occurred. In hypothyroid animals, alpha-GPD activity rose after the initial 24 h inhibition and reached levels at 72 h equal to those observed in hypothyroid rats treated with T3 only. In euthyroid animals treated with T3 and alpha-amanitin, a delayed increase in malic enzyme activity was observed at 72 h and attained values at 96 h similar to those in euthyroid animals injected with T3 only. The delayed rise in enzyme response is most easily explained by the formation of a long-lived intermediate during the exposure of the nuclear sites to T3.