Effects of iron deficiency and training on mitochondrial enzymes in skeletal muscle

We measured mitochondrial enzyme activities in skeletal muscle under conditions of iron deficiency and endurance training to assess the effects of these interventions on the contents and proportions of non-iron-containing and iron-dependent enzymes and proteins. Male Sprague-Dawley rats, 21 days of age, received a diet containing either 6 (iron deficient) or 50 mg iron/kg diet (iron sufficient). At 35 days of age animals were subdivided into sedentary and endurance training groups (running at 0.7 mph, 0% grade, 45 min/day, 6 days/wk). By 70 days of age, iron deficiency had decreased gastrocnemius muscle cytochrome c by 62% in sedentary animals. In contrast, the activities of tricarboxylic acid cycle enzymes were increased, remained unchanged or were slightly decreased, indicating that iron deficiency markedly altered mitochondrial composition. Endurance training increased cytochrome c (35%), tricarboxylic acid cycle enzymes (approximately 15%), and manganese superoxide dismutase (33%) in iron-deficient rats, whereas the same exercise regimen had no effect on the skeletal muscle of iron-sufficient animals. The interactive effect of dietary iron deficiency and mild exercise on mitochondrial enzymes suggests that adaptation to a training stimulus is, to some extent, geared to the relationship between the energy demand of exercise and the capacity for O2 transport and utilization.     

Hepatic adaptations to iron deficiency and exercise training.

Brooks et al. [Am. J. Physiol. 253 (Endocrinol. Metab. 16): E461-E466, 1987] demonstrated an elevated gluconeogenic rate in resting iron-deficient rats. Because physical exercise also imposes demand on this hepatic function, we hypothesized that exercise training superimposed on iron deficiency would augment the hepatic capacity for amino acid transamination/deamination and pyruvate carboxylation. Sprague-Dawley rats (n = 32) were obtained at weaning (21 days of age) and randomly assigned to iron-sufficient (dietary iron = 60 mg iron/kg diet) or iron-deficient (3 mg iron/kg) dietary groups. Dietary groups were subdivided into sedentary and trained subgroups. Treadmill training was 4 wk in duration, 6 days/wk, 1 h/day, 0% grade. Treadmill speed was initially 26.8 m/min and was decreased to 14.3 m/min over the 4-wk training period. The mild exercise-training regimen did not affect any measured variable in iron-sufficient rats. In contrast, in iron-deficient animals, training increased endurance capacity threefold and reduced blood lactate and the lactate-to-alanine ratio during submaximal exercise by 34 and 27%, respectively. The mitochondrial oxidative capacity of gastrocnemius muscle was increased 46% by training. However, the oxidative capacity of liver was not affected by either iron deficiency or training. Maximal rates of pyruvate carboxylation and glutamine metabolism by isolated liver mitochondria were also evaluated. Iron deficiency and training interacted to increase pyruvate carboxylation by intact mitochondria. Glutamine metabolism was increased roughly threefold by iron deficiency alone, and training amplified this effect to a ninefold increase over iron-sufficient animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity-induced adaptations in skeletal muscles of iron-deficient rabbits

The purpose of this study was to determine whether severe iron deficiency alters the adaptive response of skeletal muscle fibers to a sustained increase in tonic contractile activity. Seven weanling rabbits consumed a low iron diet and underwent phlebotomy twice weekly for 6 mo, resulting in severe anemia (mean Hb 5.5 g/dl). Compared with control animals, tibialis anterior skeletal muscles of iron-deficient animals exhibited reduced concentrations of cytochrome c (4.4 +/- 0.7 vs. 8.6 +/- 0.7 nmol/g tissue; P less than 0.01), and reduced activities of citrate synthase (83 +/- 10 vs. 133 +/- 13 mU/mg protein; P less than 0.01) and cytochrome-c oxidase (2.2 +/- 0.2 vs. 3.6 +/- 0.5 U/mg protein; P less than 0.05). In these muscles mitochondria were swollen and displayed deformed cristae. Less severe biochemical abnormalities were observed in cardiac and soleus skeletal muscles. Ten days of continuous electrical stimulation of the motor nerve supplying anterior compartment muscles of iron-deficient rabbits increased expression of mitochondrial proteins: cytochrome c was increased to 154% of control levels (P less than 0.05), and cytochrome-c oxidase and citrate synthase activities were increased to 199 and 272% of control levels, respectively (P less than 0.005). In addition, electrical pacing increased the fractional volume of mitochondria observed by electron microscopy and reduced the activity of aldolase A by 28% (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron deficiency decreases gluconeogenesis in isolated rat hepatocytes

Dietary iron deficiency in rats results in increased blood glucose turnover and recycling. We measured the rates of glucose production in isolated hepatocytes from iron-sufficient (Fe+) and iron-deficient (Fe-) rats to assess the intrinsic capacity of the Fe- liver to carry out gluconeogenesis. Low-iron and control diets were given to 21-day-old female rats. After 4-5 wk, hemoglobin concentrations averaged 4.1 g/dl in the Fe- and 14.3 g/dl in the Fe+ animals. In the hepatocytes from Fe- rats, there was a 35% decrease in the rate of glucose production from 1 mM pyruvate + 10 mM lactate, a 48% decrease from 0.1 mM pyruvate + 1 mM lactate, a 39% decrease from 1 mM alanine, and a 48% decrease from 1 mM glycerol. The addition of 5 microM norepinephrine or 0.5 microM glucagon to the incubation media produced stimulatory effects on hepatocytes from both Fe- and Fe+ rats, resulting in the maintenance of an average difference of 38% in the rates of gluconeogenesis between the two groups. Studies on isolated liver mitochondria and cytosol revealed alpha-glycerophosphate-cytochrome c reductase and phospho(enol)pyruvate carboxykinase activities to be decreased by 27% in Fe- rats. We conclude that because severe dietary iron deficiency decreases gluconeogenesis in isolated rat hepatocytes, the increased gluconeogenesis demonstrated by Fe- rats in vivo is attributable to increased availability of gluconeogenic substrates and upregulation of the pathway.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tanger?s, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.     

Caco-2 cell line: a system for studying intestinal iron transport across epithelial cell monolayers.

Iron transport across polarized intestinal epithelium was studied by using Caco-2 cells grown in bicameral chambers. When cells were grown under conditions of low, normal, or high iron concentration not only was the iron content of the cells markedly altered but the low iron cells exhibited a nearly 2-fold increase in transepithelial electrical resistance (TEER). 59Fe uptake from the apical surface into cells and transport into the basal chamber was affected both by the valency of the iron and the iron status of the cells. Uptake from 59Fe(II)-ascorbate was about 600 pmol 59Fe/h per mg protein, increased about 2-fold in low iron cells, and was about 13-200-fold greater than uptakes from 59Fe(III) chelated to nitrilotriacetic acid, BSA, or citrate. Transport into the basal chamber from 59Fe(II)-ascorbate was 3.7 +/- 1.7 pmol/h per cm2 for Fe-deficient cells vs. 0.72 +/- 0.1 pmol/h per cm2 for normal-Fe cells and from 59Fe(III)-BSA 1.1 +/- 0.2 pmol/h per cm2 vs. 0.3 +/- 0.03 pmol/h per cm2 for deficient vs. normal iron cells, respectively. The greater transport of iron both from Fe(II) and in iron deficient cells supports the use of the Caco-2 cells as a model for iron transport.     

Distribution and metabolism of iron in muscles of iron-deficient rats

Iron-deficiency anemia leads directly to both reduced hemoglobin levels and work performance in humans and experimental animals. In an attempt to observe a direct link between work performance and insufficient iron at the cellular level, we produced severe iron deficiency in female weanling Sprague-Dawley rats following five weeks on a low-iron diet. Deficient rats were compared with normal animals to observe major changes in hematological parameters, body weight, and growth of certain organs and tissues. The overall growth of iron-deficient animals was approximately 50% of normal. The ratio of organ weight: body weight increased in heart, liver, spleen, kidney, brain, and soleus muscle in response to iron deficiency. Further, mitochondria from heart and red muscle retained their iron more effectively under the stress of iron deficiency than mitochondria from liver and spleen. Metabolism of iron in normal and depleted tissue was measured using tracer amounts of⁵⁹Fe administered orally. As expected, there was greater uptake of tracer iron by iron-deficient animals. The major organ of iron accumulation was the spleen, but significant amounts of isotope were also localized in heart and brain. In all muscle tissue examined the⁵⁹Fe preferentially entered the mitochondria. Enhanced mitochondrial uptake of iron prior to any detectable change in the hemoglobin level in experimental animals may be indicative of nonhemoglobin related biochemical changes and/or decrements in work capacity.     

Does nitric oxide contribute to iron-dependent brain injury after experimental cerebral ischaemia?

Experimental and clinical data suggest that iron has a key role in cerebral ischaemia. We measure infarct volume and analyse the nitric oxide responses to brain injury in rat stroke model after increased oral iron intake. Permanent middle cerebral artery occlusion (MCAO) was performed in a group of 20 male Wistar rats, 10 of which were fed with a control diet and 10 of which were fed with iron-enriched diet containing 2.5% carbonyl iron for 9 weeks. L-arginine and nitric oxide metabolites were determined in blood samples before and at 2, 6, 8 and 48 h after MCAO. Infarct volume, thiobarbituric acid reaction substances (TBARS) and tissue iron were measured at 48 h. Infarct volume was 66% greater in the iron-fed rats than in the control group. Iron-fed animals showed significantly higher levels of TBARS. Liver iron stores (3500 +/- 199 vs 352 +/- 28 microg Fe/g, p<0.0001) but not brain iron stores (131 +/- 11 vs 139 +/- 8 microg Fe/g, p=0.617), were significantly higher in the iron-fed group. L-arginine levels were slightly lower in iron-fed rats and decreased significantly in both groups at 6 and 8 hours after MCAO. The levels of the stable end products of NOS (NOx = nitrite + nitrate) were significantly higher in iron-fed rats before MCAO (16.2 +/- 2.2 vs. 9.6 +/- 0.8 micromol x L(-1), p<0.05), with a further increase during the six first hours after MCAO in both groups. These results suggest that the iron overload that increases both superoxide and nitric oxide production leads to peroxynitrite formation, thus enhancing brain damage.     

Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins

Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tanger?s, A., Flatmark, T., B?ckstr?m, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162-175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04 +/- 0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37 degrees C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

Mitochondrial iron not bound in heme and iron-sulfur centers. Estimation, compartmentation and redox state

A method is described for the assay of total mitochondrial non-heme iron and a fraction which does not belong to the iron-sulfur proteins (FeS centers) of the outer and inner membrane. The assay of the latter fraction, which is termed 'non-heme non-FeS iron', is based on the formation of a chelate of Fe(II) with bathophenanthroline sulfonate in osmotically swollen mitochondria under conditions where the FeS centers are quite stable as determined by EPR spectroscopy at 20.4 K, 93 K and 123 K. The 'non-heme non-FeS iron', which in normal rat liver mitochondria amounts to approx. one third of the total mitochondrial iron (i.e. 1.7 +/- 0.3 nmol . mg-1 protein), does not represent a homogeneous pool of iron. Based on studies of its reaction with bathophenanthroline sulfonate and the dependency of this reaction on reducing agents in mitochondria and mitoplasts, evidence is presented that this non-heme iron is present in two major pools in which the inner membrane constitutes the barrier. A minor fraction (i.e. 0.4 +/- 0.2 nmol . mg-1 protein) is localized to the 'outer' compartment and a major fraction (i.e. 1.1 +/- 0.1 nmol . mg-1 protein) is localized to the 'inner' compartment and is equally distributed between the inner membrane and the matrix. The experiments described in this study also indicate that approximately half of the 'non-heme non-FeS iron' of the 'inner' pool is in the ferrous form in mitochondria as isolated, and this was not increased when oxidizable substrates were added to the mitochondria. Although the biological significance of this iron pool is not yet clear, it is likely that it represents a transit iron pool being the proximate iron donor for heme synthesis catalyzed by the enzyme ferrochelatase.     

锌和铁缺乏对枳生理指标、矿物质含量及叶片超微结构的影响

采用营养液培养法,研究了缺锌(0μmol·L-1 Zn2+)、缺铁(0μmol.L-1 Fe-EDTA)条件下柑橘砧木枳的生理胁迫反应.结果表明:1)锌、铁缺乏使枳生物量与根系活力均显著下降,叶片与根系中的SOD活性明显上升;叶片与根系中的POD活性在缺锌下显著增高,但在缺铁胁迫下显著降低;缺锌处理的根系CAT活性显著上升,但缺铁处理下的CAT活性与对照无显著差异.2)缺铁处理的根部K、Mg、P含量及缺锌处理的地上部K含量均显著降低;缺铁处理的根部和地上部Zn、Cu含量以及缺锌处理的根部Fe、Mn及地上部Mn含量均显著增高.3)叶肉细胞超微结构变化显著,缺铁胁迫下细胞器受损程度较重,如叶绿体、线粒体空泡化严重,叶绿体膜及类囊体片层模糊,质体小球明显增多,无淀粉粒;而缺锌处理时叶绿体基粒片层排列松散、数目明显减少,质体小球明显增多.     

Loss of mitochondrial membrane potential is associated with increase in mitochondrial volume: physiological role in neurones

Mitochondrial volume homeostasis is a housekeeping cellular function, thought to help regulate oxidative capacity, apoptosis, and mechanical signaling. The volume is mainly regulated by potassium flux into and out of the matrix and controlled by the electrochemical potential. Mitochondrial depolarization will therefore affect this flux but studies showing how have not been consistent, and it is unclear what mitochondrial volume changes also occur. The aim of the present study was to investigate mitochondrial volume changes in permeabilized neurons under various bioenergetic conditions using deconvolution confocal microscopy. Under control conditions, mitochondria in situ appeared rod-shaped with mean length, surface area, and volume values of 2.29+/-0.10 microm, 1.41+/-0.10 microm2, and 0.062+/-0.006 microm3, respectively (n=42). Valinomycin, a K+-selective ionophore, increased mitochondrial volume by 63+/-22%, although surface area was almost unchanged because mitochondrial shape became more spherical. Pinacidil, an opener of mitochondrial ATP-dependent channels, produced similar effects, although some mitochondria were insensitive to its action. Mitochondrial depolarization with the protonophore FCCP, or with respiratory chain inhibitors antimycin and sodium azide was associated with a considerable increase in mitochondrial volume (by 75%-140%). Effects of mitochondrial modulators were also studied in intact neurones. Tracking of single mitochondria showed that during 65+/-2% of their time, mitochondria were motile with an average velocity of 0.19+/-0.01 microm/s. Antimycin, azide, and FCCP induced mitochondrial swelling and significantly decreased mitochondrial motility. In the presence of pinacidil, swollen mitochondria had reduced their motility, although mitochondria with normal volume stayed motile. These data show that mitochondrial depolarization was followed by significant swelling, which, in turn, impaired mitochondrial trafficking.     

Proinflammatory cytokines promote glial heme oxygenase-1 expression and mitochondrial iron deposition: implications for multiple sclerosis

Proinflammatory cytokines, pathological iron deposition, and oxidative stress have been implicated in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). HO-1 mRNA levels and mitochondrial uptake of [(55)Fe]Cl(3)-derived iron were measured in rat astroglial cultures exposed to interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) alone or in combination with the heme oxygenase-1 (HO-1) inhibitors, tin mesoporphyrin (SnMP) or dexamthasone (DEX), or interferon beta1b (INF-beta). HO-1 expression in astrocytes was evaluated by immunohistochemical staining of spinal cord tissue derived from MS and control subjects. IL-1beta or TNF-alpha promoted sequestration of non-transferrin-derived (55)Fe by astroglial mitochondria. HO-1 inhibitors, mitochondrial permeability transition pore (MTP) blockers and antioxidants significantly attenuated cytokine-related mitochondrial iron sequestration in these cells. IFN-beta decreased HO-1 expression and mitochondrial iron sequestration in IL-1beta- and TNF-alpha-challenged astroglia. The percentage of astrocytes coexpressing HO-1 in affected spinal cord from MS patients (57.3% +/- 12.8%) was significantly greater (p < 0.05) than in normal spinal cord derived from controls subjects (15.4% +/- 8.4%). HO-1 is over-expressed in MS spinal cord astroglia and may promote mitochondrial iron deposition in MS plaques. In MS, IFN-beta may attenuate glial HO-1 gene induction and aberrant mitochondrial iron deposition accruing from exposure to proinflammatory cytokines.     

Three-Dimensional Reconstruction,by TEM Tomography,of the Ultrastructural Modifications Occurring in Cucumis sativus L. Mitochondria under Fe Deficiency

Background

Mitochondria, as recently suggested, might be involved in iron sensing and signalling pathways in plant cells. For a better understanding of the role of these organelles in mediating the Fe deficiency responses in plant cells, it is crucial to provide a full overview of their modifications occurring under Fe-limited conditions. The aim of this work is to characterize the ultrastructural as well as the biochemical changes occurring in leaf mitochondria of cucumber (Cucumis sativus L.) plants grown under Fe deficiency.

Methodology/Results

Mitochondrial ultrastructure was investigated by transmission electron microscopy (TEM) and electron tomography techniques, which allowed a three-dimensional (3D) reconstruction of cellular structures. These analyses reveal that mitochondria isolated from cucumber leaves appear in the cristae junction model conformation and that Fe deficiency strongly alters both the number and the volume of cristae. The ultrastructural changes observed in mitochondria isolated from Fe-deficient leaves reflect a metabolic status characterized by a respiratory chain operating at a lower rate (orthodox-like conformation) with respect to mitochondria from control leaves.

Conclusions

To our knowledge, this is the first report showing a 3D reconstruction of plant mitochondria. Furthermore, these results suggest that a detailed characterization of the link between changes in the ultrastructure and functionality of mitochondria during different nutritional conditions, can provide a successful approach to understand the role of these organelles in the plant response to Fe deficiency.     

Mitochondrial iron loss from leukemia cells injured by macrophages. A possible mechanism for electron transport chain defects

Activated macrophages inhibit replication of murine lymphoblastic leukemia L1210 cells without lysis. This inhibition of replication is associated with abnormalities of mitochondrial electron transport at the level of NADH dehydrogenase (NADH-DH) and succinate dehydrogenase (SDH). The mechanism of inhibition is unknown, although it has been demonstrated that as NADH-DH and SDH activity is lost, iron is released from cells. Because both NADH-DH and SDH contain numerous iron-sulfur clusters, damage to these structures may be one result of injury by activated macrophages. L1210 cells were labeled with 55Fe and co-cultivated with activated murine peritoneal macrophages (injured L1210 cells). At 48 h, injured L1210 cells had released 83 +/- 8% (mean +/- SEM of 55Fe activity into the media, compared with 25 +/- 4% release from control and 37 +/- 7% from nondividing mitomycin C-treated control cells. All cells were greater than 90% viable. These differences were also reflected in the iron content of the cells. Mitochondria were then separated by centrifugation after cell disruption and 55Fe activity was found to be similarly decreased in both mitochondrial and nonmitochondrial fractions of injured L1210 cells. To further characterize the changes in mitochondrial iron content, mitochondrial proteins from injured and control L1210 cells were separated by IEF and 55Fe activity of gel slices was determined. There was selective loss of 55Fe activity in the area of the gel corresponding to SDH and NADH-DH, suggesting that iron loss from iron-sulfur clusters may occur in L1210 cells injured by activated macrophages. Iron uptake into L1210 cells after removal from macrophages showed a rapid large influx of radioactive iron. L1210 cells in contact with macrophages appear to develop an iron-depleted state, which is dependent on the continued presence of macrophages.     

Protection of rats by clofibrate against the hypoglycaemic and toxic effects of hypoglycin and pent-4-enoate. An ultrastructural and biochemical study.

An ultrastructural and biochemical study of the toxic and hypoglycaemic effects of hypoglycin and pent-4-enoate was made on the livers of normal and clofibrate-fed rats. Injection of hypoglycin to rats doubles (from 22% to 44%) the volume fraction of mitochondria and decreases (from 1.05% to 0.26%) the volume fraction of peroxisomes in hepatocytes. The fast-acting toxin pent-4-enoate causes few ultrastructural changes except for the accumulation of lipids. In male adult rats fed with 0.5% clofibrate in their diet for 1-2 months, the volume fraction occupied by peroxisomes and mitochondria in hepatocytes rose to 6.26% and 29.5% respectively. Clofibrate feeding apparently protected the animals against the toxic, hypoglycaemic and hypothermic effects of hypoglycin and of pent-4-enoate, and completely prevented the ultrastructural damage caused by hypoglycin. After hypoglycin administration, hepatic mitochondrial butyryl-CoA dehydrogenase activity was inhibited by more than 90% and, surprisingly, the activity of the peroxisomal enzymes studied was largely preserved. When hypoglycin was given to rats fed on a clofibrate-containing diet, the oxidation of decanoylcarnitine, which was incomplete after hypoglycin treatment alone, remained incomplete with uncoupled mitochondria, but became apparently complete with coupled mitochondria. In the latter condition, there was a slowing of the rate during the last quarter of the pulse of oxygen uptake. Further, butyryl-CoA dehydrogenase activity was much less affected by hypoglycin in clofibrate-fed animals. Pent-4-enoate does not inhibit beta-oxidation in coupled mitochondria from clofibrate-treated rats.     

Riboflavin-binding protein. Concentration and fractional saturation in chicken eggs as a function of dietary riboflavin.

In female rats with porphyria induced by hexachlorobenzene, the amounts of non-haem iron and porphyrins in liver mitochondrial fractions were increased almost 3-fold and greater than 500-fold respectively compared with that of untreated animals. A considerable fraction of both iron and porphyrins in this fraction was shown to be located in lysosomes. Thus mitochondrial preparations, which were further depleted of lysosomes by Percoll-density-gradient centrifugation, contained 2.78 +/- 0.75 and 2.99 +/- 0.49 nmol of non-haem iron/mg of protein when isolated from the liver of control rats and hexachlorobenzene-treated rats respectively. Mitochondria isolated from the liver of hexachlorobenzene-treated animals contained a pool of iron (about 1 nmol/mg of protein) that was available for haem synthesis in vitro. This pool is similar to that previously reported for mitochondria isolated from the liver of rats with normal haem synthesis. Hexachlorobenzene treatment, therefore, does not affect the iron status of the mitochondria.     

Long-term trophic effect of ACTH on rat adrenocortical cells

Summary The changes occurring in rat adrenocortical cells (zona fasciculata) during an 8 day period of treatment with ACTH, were investigated by morphometric and autoradiographic methods.The most important ultrastructural change consists in a conspicuous increase in the smooth endoplasmic reticulum, that accounts for about 50% of the total increase of cellular volume. Also the mitochondrial fraction shows a significant increase, which is found to be due both to the increment in the number of mitochondria per cell and to the increase in the mean volume of organelles themselves.The quantitative autoradiographic data, indicating an increment in the incorporation of 3H-orotate and 3H-leucine into adrenocortical cells of the treated animals, allow us to conclude that the ACTH-induced ultrastructural changes are the morphological expression of a stimulation of the cellular protein synthesis.Since mitochondria are largely autonomous in the synthesis of their enzymes and structural proteins, it is possible to hypothesize that ACTH also intervenes in the regulation of the mitochondrial protein synthesis.The authors wish to express their sincere appreciation to Mr. G. Gottardo for his excellent technical assistance.