M-aconitase Activity Research Articles

Abstract 5750: Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer

Abstract Introduction: Treatment choices for early-stage prostate cancer include mostly watchful waiting, radical prostatectomy, or radiation therapy, whereas for advanced cases hormone therapy followed by chemo/radiotherapy are the preferred options, despite their notable side effects. There is a need for more effective therapy with lower side effects, especially for advanced scenarios. Unlike normal epithelial prostate cells, cancerous ones show reduced zinc (Zn) levels due to ZIP1 zinc transporter downregulation. Zn deficiency allows m-aconitase to remain active, making prostate cancer cells energy efficient and grow. High levels of Zn are needed to inhibit m-aconitase, the enzyme responsible for the first reaction in the Krebs cycle. This study aims to induce energy starvation and cell death of prostate cancer cells by inhibiting m-aconitase using the prostate-specific membrane antigen (PSMA)-targeting liposomal zinc. Methods: Liposomes were prepared by standard reverse phase evaporation method with DSPC, Cholesterol and DSPE-mPEG(2000) lipids. Zinc chloride was used to load Zn+2 into liposome following passive loading method. PSMA-expressing (PC3-PIP) and PSMA-null (PC3-LUC) human prostate cancer cells were used for in vitro studies. In the cell uptake study, cells were incubated with Dil-dye labeled liposomes for 2 hours before analyzing them by microscopy and flow cytometry. For the uptake inhibition study, cells were pre-incubated with PSMA-specific peptide for 2 hours before incubation with liposomes. Cytotoxicity studies were performed with PSMA-targeting Zn-loaded liposomes in PC3-PIP cells. Results: We conjugated the PSMA-specific peptide and control peptide (ASP3) with DSPE-PEG(3400)-NHS, and used them at various amounts (1 to 4 mol% of total lipid) to prepare Zn-loaded PSMA-targeting liposomes (TL) and control liposomes (CL), respectively. The average hydrodynamic diameter of all the liposomes was 120 to 130 nm, and zeta potential ranged from -16 to -12 mV. The encapsulation efficiency and loading capacity of Zn+2 were 0.6% and 8.5%, respectively. Cellular uptake studies showed a higher accumulation of PSMA-targeting liposome (TL) compared to non-targeting liposome (CL) in PSMA-expressing PC3 cells, whereas both liposomes showed low uptake in PSMA non-expressing cell. Furthermore, pre-treatment of PSMA-expressing cells with PSMA-specific peptide reduced the uptake of TL, indicating that the uptake of TL liposome is PSMA dependent. Finally, cytotoxicity studies showed that Zn-loaded TL, induces cell death in the PSMA-expressing cells. Overall, we developed PSMA-targeting Zn-containing liposomes that selectively target and kill PSMA-expressing prostate cancer cells. Future work includes testing the effect of Zn-loaded TL on m-aconitase activity and testing of in vivo efficacy of the formulation in prostate cancer models. Citation Format: Sujan K. Mondal, Alexander L. Klibanov, Anna Moore. Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5750.

Antiproliferative effects of zinc-citrate compound on hormone refractory prostate cancer

To investigate the antiproliferative effects of zinc-citrate compound on hormone refractory prostate cancer (HRPC). HRPC cell line (DU145) and normal prostate cell line (RWPE-1) were treated with zinc, citrate and zinc-citrate compound at different time intervals and concentrations to investigate the effect of zinc-citrate compound. Mitochondrial (m)-aconitase activity was determined using aconitase assay. DNA laddering analysis was performed to investigate apoptosis of DU145 cells. Molecular mechanism of apoptosis was investigated by Western blot analysis of P53, P21(waf1), Bcl-2, Bcl-xL and Bax, and also caspase-3 activity analysis. Treatment with zinc-citrate compound resulted in a time- and dose-dependent decrease in cell number of DU145 cells in comparison with RWPE-1. M-aconitase activity was significantly decreased. DNA laddering analysis indicated apoptosis of DU145 cells. Zinc-citrate compound increased the expression of P21(waf1) and P53, and reduced the expression of Bcl-2 and Bcl-xL proteins but induced the expression of Bax protein. Zinc-citrate compound induced apoptosis of DU145 cells by activation of the caspase-3 pathway. Zinc-citrate compound can induce apoptotic cell death in DU145, by caspase-3 activation through up-regulation of apoptotic proteins and down-regulation of antiapoptotic proteins.

Induction of Apoptosis of Bladder Cancer Cells by Zinc-Citrate Compound

PurposeZinc is one of the trace minerals in the body and is known to have an anticancer effect by inducing apoptosis in prostate cancer. We aimed to investigate the antiproliferative effects of a zinc-citrate compound in bladder cancer.Materials and MethodsA bladder cancer cell line (MBT-2) was treated with a zinc-citrate compound at different time intervals and concentrations. Mitochondrial (m)-aconitase activity was determined by use of the aconitase assay. DNA laddering analysis was performed to investigate apoptosis of MBT-2 cells. The molecular mechanism of apoptosis was investigated by Western blot analysis of p53, p21waf1, Bcl-2, Bcl-xL, and Bax and also by caspase-3 activity analysis.ResultsTreatment with the zinc-citrate compound resulted in a time- and dose-dependent decrease in cell number of MBT-2 cells. M-aconitase activity was significantly decreased. DNA laddering analysis indicated apoptosis of MBT-2 cells. The zinc-citrate compound increased the expression of p21waf1 and p53 and reduced the expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. The zinc-citrate compound induced apoptosis of MBT-2 cells by activation of the caspase-3 pathway.ConclusionsWe have shown that a zinc-citrate compound induces apoptotic cell death in a bladder cancer cell line, MBT-2, by caspase-3 activation through up-regulation of apoptotic proteins and down-regulation of antiapoptotic proteins.

Effect of metabolic inhibitors on ATP and citrate content in PC3 prostate cancer cells

In normal prostate epithelial cells low m-aconitase activity decreases citrate oxidation leading to citrate accumulation. In prostate cancer cells m-aconitase activity is increased and citrate content is lower. The effect of inhibition of m-aconitase on ATP production by prostate cancer cells (PC3) is not known nor is the contribution of glycolysis versus respiration. ATP content of PC3 cells as affected by inhibition of m-aconitase (fluoroacetate (FA), zinc), inhibition of glycolysis (2DxG), or respiration (DNP, oligomycin) was determined. The ability to maintain ATP using glucose or glutamine as sole substrate was also determined. Intermediates including ATP, lactate, glucose, and glutamine were assayed in neutralized perchloric acid (PCA) cell extracts, virgin, and conditioned medium by enzymatic fluorometry. Data show that inhibition of m-aconitase, glycolysis, or respiration alone did not decrease ATP content. Inhibition of both glycolysis and respiration were required to decrease ATP content. PC3 cells were able to produce ATP with either glucose or glutamine as sole substrate. Though FA clearly inhibited m-aconitase there was no evidence that zinc had a similar effect. PC3 cells can support ATP production when m-aconitase is inhibited by using glycolysis or oxidation of substrate (e.g., glutamine) entering the TCA cycle distal to citrate.

Zinc as an anti-tumor agent in prostate cancer and in other cancers

Human prostate glandular epithelial cells have the unique capability of accumulating high levels of zinc. This is essential to inhibit m-aconitase activity so that citrate can accumulate for secretion into prostatic fluid, which is a major function of the prostate gland. As a result, the Krebs cycle is truncated with the consequence of the lost ATP production that would result from citrate oxidation. The cellular accumulation of zinc also inhibits mitochondrial terminal oxidation and respiration. In addition to these metabolic effects, zinc accumulation exhibits anti-proliferative effects via its induction of mitochondrial apoptogenesis. Zinc accumulation also inhibits the invasive/migration activities in malignant prostate cells. The anti-proliferative effects and the effects on invasion and migration occur through zinc activation of specific intracellular signaling pathways. Consequently, these effects impose anti-tumor actions by zinc. The ability of prostate cells to accumulate zinc is due to the expression and activity of the zinc uptake transporter, ZIP1. To avoid the anti-tumor effects of zinc, in prostate cancer the malignant prostate cells exhibit a silencing of ZIP1 gene expression accompanied by a depletion of cellular zinc. Therefore we regard ZIP1 as a tumor suppressor gene in prostate cancer. In addition to prostate cells, similar tumor suppressor effects of zinc have been identified in several other types of tumors.

Roles of the Mammalian Cytosolic Cysteine Desulfurase, ISCS, and Scaffold Protein, ISCU, in Iron-Sulfur Cluster Assembly

Iron-sulfur clusters are prosthetic groups composed of sulfur and iron that are found in respiratory chain complexes and numerous enzymes. Iron-sulfur clusters are synthesized in a multistep process that utilizes cysteine desulfurases, scaffold proteins, chaperones, and iron donors. Assembly of iron-sulfur clusters occurs in the mitochondrial matrix of mammalian cells, but cytosolic isoforms of three major mammalian iron-sulfur cluster (ISC) assembly components have been found, raising the possibility that de novo iron-sulfur cluster biogenesis also occurs in cytosol. The human cysteine desulfurase, ISCS, has two isoforms, one of which targets to the mitochondria, whereas the other less abundant form is cytosolic and nuclear. The open-reading frame of cytosolic mammalian ISCS begins at the second AUG of the transcript and lacks mitochondrial targeting information. Yeast complementation experiments have suggested that the human cytosolic ISCS isoform (c-ISCS) cannot be functional. To evaluate function of c-ISCS, we overexpressed the human cytosolic ISCS in yeast Pichia pastoris and showed that the cytosolic form of ISCS is an active cysteine desulfurase that covalently binds 35S acquired from desulfuration of radiolabeled cysteine. Human cytosolic ISCS dimerized as efficiently as bacterial ISCS and formed a complex in vitro with overexpressed cytosolic human ISCU. When incubated with iron regulatory protein 1, cysteine, and iron, the cytosolic forms of ISCS and ISCU facilitated efficient formation of a [4Fe-4S] cluster on IRP1. Thus, the cytosolic form of ISCS is a functional cysteine desulfurase that can collaborate with cytosolic ISCU to promote de novo iron-sulfur cluster formation.

Antiproliferative and apoptotic effects of zinc–citrate compound (CIZAR®) on human epithelial ovarian cancer cell line, OVCAR-3

Objective. Zinc inhibits the growth of several carcinoma cells through induction of cell cycle arrest and apoptosis. The intracellular concentration of zinc and its dynamic changes are critically important in cell biology. We investigated the effects of zinc–citrate compound (CIZAR®) on normal human ovarian epithelial cells (NOSE) and human epithelial ovarian cancer cell line, OVCAR-3. Methods. To investigate the potential effect of CIZAR® on cell growth and survival, cells were treated with different doses and exposed to different times. Intracellular concentration of zinc was measured by colorimetric assay. Mitochondrial aconitase activity was determined in cell extracts using aconitase assay. The flow cytometric assay, DNA laddering, and morphological analysis were done to investigate cytotoxic effects of CIZAR®. Molecular mechanism of cell death was investigated by p53, Bcl-xL, Bcl-2, Bax protein, activity of caspase-3 and -12, and activity of telomerase. Results. CIZAR®-induced zinc accumulation in OVCAR-3 cells was higher than that in NOSE cells. CIZAR® treatment resulted in a time- and dose-dependent decrease in cell number in OVCAR-3 cells in comparison with NOSE cells. M-aconitase activity was significantly decreased in OVCAR-3 cells within 4 h exposure to CIZAR® but relatively constant in NOSE cells. The flow cytometric assay, DNA laddering, and morphological analysis indicated apoptosis in OVCAR-3 cells but not in NOSE cells. CIZAR® increased the expression of p21 waf1 which is a part of p53-independent pathway and induced reduction of telomerase activity. CIZAR® reduced expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. CIZAR® induced apoptosis of OVCAR-3 cells by activation of caspase-12 and caspase-3 pathway. Conclusions. Exposure to CIZAR® induces apoptosis in OVCAR-3 cells which accumulate high intracellular levels of zinc, but not in NOSE cells, which do not accumulate high levels of zinc. CIZAR® prevents the proliferation of OVCAR-3 cells by inactivation of m-aconitase activity and induces apoptosis by induction of proapoptotic gene (Bax), repression of antiapoptotic genes (Bcl-2, Bcl-xL), and consequently activation of caspase-3. CIZAR® also induced activation of caspase-12. The CIZAR® will offer new window in prevention and treatment of epithelial ovarian cancer.

Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues

BackgroundIn prostate cancer, normal citrate-producing glandular secretory epithelial cells undergo a metabolic transformation to malignant citrate-oxidizing cells. m-Aconitase is the critical step involved in this altered citrate metabolism that is essential to prostate malignancy. The limiting m-aconitase activity in prostate epithelial cells could be the result of a decreased level of m-aconitase enzyme and/or the inhibition of existing m-aconitase. Earlier studies identified zinc as an inhibitor of m-aconitase activity in prostate cells; and that the depletion of zinc in malignant cells is an important factor in this metabolic transformation. However, a possibility remains that an altered expression and level of m-aconitase enzyme might also be involved in this metabolic transformation. To address this issue, the in situ level of m-aconitase enzyme was determined by immunohistochemical analysis of prostate cancer tissue sections and malignant prostate cell lines.ResultsThe immunocytochemical procedure successfully identified the presence of m-aconitase localized in the mitochondrial compartment in PC-3, LNCaP, and DU-145 malignant prostate cell lines. The examination of prostate tissue sections from prostate cancer subjects demonstrated that m-aconitase enzyme is present in the glandular epithelium of normal glands, hyperplastic glands, adenocrcinomatous glands, and prostatic intraepithelial neoplastic foci. Quantitative analysis of the relative level of m-aconitase in the glandular epithelium of citrate-producing adenomatous glands versus the citrate-oxidizing adenocarcinomatous glands revealed no significant difference in m-aconitase enzyme levels. This is in contrast to the down-regulation of ZIP1 zinc transporter in the malignant glands versus hyperplastic glands that exists in the same tissue samples.ConclusionThe results demonstrate the existence of m-aconitase enzyme in the citrate-producing glandular epithelial cells; so that deficient m-aconitase enzyme is not associated with the limiting m-aconitase activity that prevents citrate oxidation in these cells. The level of m-aconitase is maintained in the malignant cells; so that an altered enzyme level is not associated with the increased m-aconitase activity. Consequently, the elevated zinc level that inhibits m-aconitase enzyme is responsible for the impaired citrate oxidation in normal and hyperplastic prostate glandular epithelial cells. Moreover, the down-regulation of ZIP1 zinc transporter and corresponding depletion of zinc results in the increase in the activity of the existing m-aconitase activity in the malignant prostate cells. The studies now define the mechanism for the metabolic transformation that characterizes the essential transition of normal citrate-producing epithelial cells to malignant citrate-oxidizing cells.

Mitochondrial Cysteine Desulfurase Iron-Sulfur Cluster S and Aconitase Are Post-transcriptionally Regulated by Dietary Iron in Skeletal Muscle of Rats

Cysteine desulfurase IscS is required for cellular iron-sulfur protein maturation in eukaryotes and prokaryotes. In this study, we examined the effect of dietary iron intake on the expression in rat skeletal muscle of IscS in relation to 2 iron-sulfur proteins, cytosolic aconitase (c-aconitase) and mitochondrial aconitase (m-aconitase). Three groups of male weanling Wistar rats were used; 1 group was fed an iron-deficient diet (D), and the other 2 groups were pair-fed (P) or freely fed (C) a control (35 mg Fe/kg diet) diet for 1 or 2 wk. At the end of wk 1 and 2, the mitochondrial IscS protein levels in the skeletal muscle of iron-deficient rats had decreased to 45 and 50% of those of the control and pair-fed rats, respectively, whereas the IscS mRNA levels did not differ among the 3 groups, indicating that iron deficiency affected the expression of IscS protein at the post-transcriptional level. Iron deficiency caused a 55-76% reduction in c-aconitase activity and an approximately 50% reduction in the c-aconitase protein level. The m-aconitase activity and protein level in iron-deficient rats also declined to 50 and 58-64% of the control levels, respectively. Our results indicate that dietary iron modulates mitochondrial IscS protein and aconitase at the post-transcriptional level, and mitochondrial IscS may be associated with this regulation of aconitase in skeletal muscle.

Effects of potassium‐magnesium citrate supplementation on cytosolic ATP citrate lyase and mitochondrial aconitase activity in leukocytes: A window on renal citrate metabolism

An increase in urinary citrate excretion is associated with a decrease in activity of renal cortical cytosolic ATP citrate lyase (ACL) and mitochondrial aconitase (m-aconitase). Because potassium-magnesium citrate causes an increase in urinary citrate excretion, we decided to assess its effects on ACL and m-aconitase in the leukocytes of renal stone patients. Twenty male renal stone patients were supplemented with potassium-magnesium citrate twice daily (i.e. 42 mEq potassium, 21 mEq magnesium, and 63 mEq citrate per day) for a period of 1 month. Two 24-h urine and one 15-mL heparinized blood samples were collected from each patient before and after supplementation. Urine samples were analyzed for relevant biochemical compositions. Leukocytes were separated from blood samples by centrifugation and assayed for ACL and m-aconitase activity. Supplementation with potassium-magnesium citrate significantly increased urinary pH (P < 0.005) and excretions of potassium (P < 0.001), magnesium (P < 0.001) and citrate (P < 0.0001). The activity of both ACL and m-aconitase were significantly decreased (P < 0.004 and P < 0.02 respectively). The decrease in ACL activity was inversely correlated with an increase in urinary excretion of both potassium (r = -0.620, P < 0.0001) and citrate (r = -0.451, P < 0.004). A similar inverse correlation was observed between m-aconitase activity and urinary excretion of citrate (r = -0.322, P < 0.043). Changes in enzyme activity, related to citrate metabolism in leukocytes, might reflect the status of renal tubular cells.

Chronic Marginal Iron Intakes during Early Development in Mice Result in Persistent Changes in Dopamine Metabolism and Myelin Composition

Marginal iron (Fe) deficiency is prevalent in children worldwide, yet the behavioral and biochemical effects of chronic marginal Fe intakes during early development are not well characterized. Using a murine model, previous work in our laboratory demonstrated persistent behavioral disturbances as a consequence of marginal Fe intakes during early development. In the present study, Swiss-Webster mice fed a control Fe diet (75 microgram Fe/g diet, n = 13 litters) or marginal Fe diet (14 microgram Fe/g diet, n = 16 litters) during gestation and through postnatal day (PND) 75 were killed on PND 75 for assessment of tissue mineral concentrations, dopamine metabolism, myelin fatty acid composition, and c- and m-aconitase activities. In addition, these outcomes were assessed in a group of offspring (n = 13 litters) fed a marginal Fe diet during gestation and lactation and then fed a control diet from PND 21-75. Marginal Fe mice demonstrated significant differences in brain iron concentrations, dopamine metabolism and myelin fatty acid composition relative to control mice; however, no difference in c- or m-aconitase activity was demonstrated in the brain. The postnatal consumption of Fe-adequate diets among marginal Fe offspring did not fully reverse all of the observed biochemical disturbances. This study demonstrates that chronic marginal Fe intakes during early development can result in significant changes in brain biochemistry. The persistence of some of these biochemical changes after postnatal Fe supplementation suggests that they are an irreversible consequence of developmental Fe restriction.

Mitochondrial aconitase gene expression is regulated by testosterone and prolactin in prostate epithelial cells .

m-aconitase catalyzes the first step leading to the oxidation of citrate via the Krebs cycle. It is a constituitive enzyme in virtually all mammalian cells, found in excess, and is considered to be a regulatory or regulated enzyme. In contrast to these general relationships, prostate secretory epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase which minimizes the oxidation of citrate. This permits the unique prostate function of accumulating and secreting extraordinarily high levels of citrate. Previous animal studies demonstrated that testosterone and prolactin regulate the level of m-aconitase specifically in citrate-producing prostate cells. The present studies were conducted to determine if testosterone and prolactin regulated the expression of the m-aconitase gene in prostate cells, and to determine the effect of the hormones on human prostate cells. The studies were conducted with freshly prepared rat ventral, rat lateral, and pig prostate epithelial cells, and with the human malignant cell lines LNCaP and PC-3. The effects of 1 nM testosterone and 3 nM prolactin on the level of m-aconitase mRNA and on the transcription rate of m-aconitase were determined. The studies revealed that both prolactin and testosterone increase the levels of m-aconitase mRNA and the transcription rates of m-aconitase in rat ventral prostate cells, pig prostate cells, and human malignant prostate cells (LNCaP and PC-3). In contrast, both hormones decreased the level of m-aconitase mRNA and repressed m-aconitase gene transcription in rat lateral prostate cells. The hormonal regulation of m-aconitase corresponded with the levels of m-aconitase enzyme, m-aconitase activity, and citrate oxidation. In addition to the constitutive expression of m-aconitase, the m-aconitase gene is testosterone- and prolactin-regulated in specifically targeted prostate cells. The hormonal regulation of m-aconitase gene expression and biosynthesis of m-aconitase provide a regulatory mechanism for the oxidation of citrate, and consequently, the level of net citrate production by prostate. The hormonally increased expression and biosynthesis of m-aconitase in human malignant cells might be involved in the increased citrate oxidation associated with the development of true malignant cells in prostate cancer.

Zinc causes a shift toward citrate at equilibrium of the m-aconitase reaction of prostate mitochondria

Prostate secretory epithelial cells have the unique function and capability of accumulating and secreting extraordinarily high levels of citrate. To achieve this, these cells possess a uniquely limiting mitochondrial (m)-aconitase activity that minimizes the oxidation of citrate via the Krebs cycle. The steady-state citrate/isocitrate ratio of mammalian tissues is generally maintained at about 10–11/1, independent of the concentration of citrate, which is the result of the chemical equilibrium reached in the presence of m-aconitase. In contrast, the citrate/isocitrate ratio of prostate tissue is about 30–40/1. Zinc, which is also accumulated in prostate cells at much higher levels than in other cells, inhibits m-aconitase activity thereby minimizing citrate oxidation. This current report is concerned with an effect of zinc on the equilibrium of the reaction catalyzed by m-aconitase. Studies were conducted with mitochondrial extract preparations from rat ventral prostate epithelial cells. With citrate as the initial substrate, the addition of zinc (7–10 μM) to the prostate mitochondrial preparation resulted in a change in the citrate/isocitrate ratio at equilibrium from an average of 10.5/1 to 13.5/1. In contrast, the identical treatment of kidney mitochondrial preparations resulted in no zinc-induced change in the citrate/isocitrate ratio. When either cis-aconitate or isocitrate was employed as the initial substrate, the addition of zinc did not alter the citrate/isocitrate ratio of prostate or kidney preparations. Partial purification of the prostate preparation revealed that the prostate mitochondrial extract contained a putative protein (which we have designated as ‘citrate factor protein’) that is required for the zinc-induced increase in the citrate/isocitrate ratio. This novel effect of zinc provides another mechanism by which it is assured that the accumulation of citrate is maximized in citrate-producing prostate epithelial cells.

Renal cortical mitochondrial aconitase is regulated in hypo- and hypercitraturia

Chronic metabolic acidosis and K+ deficiency increase, while alkali feeding decreases proximal tubule citrate absorption and metabolism. The present studies examined the regulation of mitochondrial aconitase (m-aconitase), the first step in mitochondrial citrate metabolism, in these conditions. Rats were fed appropriate diets, and m-aconitase activity and protein abundance measured. In chronic metabolic acidosis and chronic K+ deficiency, renal cortical m-aconitase activity was increased 17% and 43%, respectively. This was associated with respective 90% and 221% increases in renal cortical m-aconitase protein abundance. With chronic alkali feeding, there was a 12% decrease in renal cortical m-aconitase activity, associated with a 35% decrease in m-aconitase protein abundance. Hepatic m-aconitase activity was not regulated in a similar manner. There was no regulation of citrate synthase, the enzyme responsible for mitochondrial citrate synthesis. These studies demonstrate tissue specific chronic regulation of renal cortical m-aconitase activity and protein abundance, which likely contributes to the hypocitraturia and hypercitraturia seen in these conditions. As m-aconitase is the only step in citrate transport and metabolism found to be regulated in alkali feeding, its regulation likely plays a significant role in mediating the hypercitraturia seen in this condition.

Zinc Inhibition of Mitochondrial Aconitase and Its Importance in Citrate Metabolism of Prostate Epithelial Cells

Prostate epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase activity that minimizes their ability to oxidize citrate. These cells also possess uniquely high cellular and mitochondrial zinc levels. Correlations among zinc, citrate, and m-aconitase in prostate indicated that zinc might be an inhibitor of prostate m-aconitase activity and citrate oxidation. The present studies reveal that zinc at near physiological levels inhibited m-aconitase activity of mitochondrial sonicate preparations obtained from rat ventral prostate epithelial cells. Corresponding studies conducted with mitochondrial sonicates of rat kidney cells revealed that zinc also inhibited the kidney m-aconitase activity. However the inhibitory effect of zinc was more sensitive with the prostate m-aconitase activity. Zinc inhibition fit the competitive inhibitor model. The inhibitory effect of zinc occurred only with citrate as substrate and was specific for the citrate --> cis-aconitate reaction. Other cations (Ca2+, Mn2+, Cd2+) did not result in the inhibitory effects obtained with zinc. The presence of endogenous zinc inhibited the m-aconitase activity of the prostate mitochondrial preparations. Kidney preparations that contain lower endogenous zinc levels exhibited no endogenous inhibition of m-aconitase activity. Studies with pig prostate and seminal vesicle mitochondrial preparations also revealed that zinc was a competitive inhibitor against citrate of m-aconitase activity. The effects of zinc on purified beef heart m-aconitase verified the competitive inhibitor action of zinc. In contrast, zinc had no inhibitory effect on purified cytosolic aconitase. These studies reveal for the first time that zinc is a specific inhibitor of m-aconitase of mammalian cells. In prostate epithelial cells, in situ mitochondrial zinc levels inhibit m-aconitase activity, which provides a mechanism by which citrate oxidation is limited.

Testosterone and prolactin stimulation ofmitochondrial aconitase in pig prostate epithelial cells

Objectives. The function of the prostate gland in many animals, including humans, is to accumulate andsecrete large quantities of citrate. This function derives from the metabolic characteristics of the prostate secretory epithelial cells. These cells possess a uniquely limiting mitochondrial aconitase (m-aconitase) that minimizes citrate oxidation and thus permits citrate to accumulate. Unfortunately, the characteristics of prostate m-aconitase and its manner of regulation have not been established. The hormones testosterone and prolactin, however, are significantly involved in regulating prostate citrate production. Thus it is reasonable to hypothesize that these hormones may be involved in the regulation of both m-aconitase and citrate oxidation. Methods. Using freshly prepared pig prostate epithelial cells, we attempted to determine the effects oftestosterone and prolactin treatment on the level of m-aconitase enzyme, on the level of m-aconitase activity, and on citrate utilization. The epithelial cells were incubated for 3 hours with either testosterone (10 −9 M), prolactin (1 μg/mL), or vehicle (control). Results. Both hormone applications caused a marked increase in the level of m-aconitase. In contrast, neitherhormone had any effect on the m-aconitase level of pig seminal vesicle cells, which are also citrate-producing cells. Moreover, neither hormone had any effect on pyruvate dehydrogenase E1a. These findings suggest that testosterone and prolactin regulation of prostate m-aconitase is a highly specific effect. Along with the increase in the level of m-aconitase enzyme, both hormones also increased m-aconitase activity and prostate-cell utilization of citrate. Conclusions. These studies demonstrate that testosterone and prolactin can regulate m-aconitase and subsequent citrate oxidation of specific prostate epithelial cells. This unique aconitase relationship is not observed in other mammalian cells.

Testosterone stimulates the biosynthesis of m-aconitase and citrate oxidation in prostate epithelial cells

Mitochondria (m-)aconitase is a rate-limiting regulatory enzyme in prostate epithelial cells which minimizes citrate oxidation by these cells. This unique metabolic characteristic is responsible for the ability of the prostate to accumulate and secrete extraordinarily high levels of citrate. Testosterone is a major regulator of prostate growth and function, and stimulates citrate oxidation. Therefore, an important action of testosterone might be its stimulation of m-aconitase in prostate epithelial cells. Studies were conducted with rat ventral prostate (VP) epithelial cells to establish the effect of testosterone on the level of m-aconitase and corresponding citrate oxidation. Physiological concentrations (10 −7–10 −10 M) of testosterone in vitro markedly increased the level of m-aconitase in freshly prepared isolated prostate epithelial cells. This increase was apparent within 3 h of exposure to the hormone. The stimulatory effect of testosterone on m-aconitase was abolished by actinomycin D and by cycloheximide. Both the level of m-aconitase enzyme and the level of m-aconitase activity were similarly increased by testosterone treatment. Correspondingly, testosterone increased the rate of mitochondrial citrate oxidation while having no effect on the rate of isocitrate oxidation, thereby demonstrating that the action of testosterone is specifically targeted at the m-aconitase reaction. In vivo studies revealed that castration markedly decreased and testosterone administration increased the m-aconitase level of prostate epithelial cells. In contrast, neither liver nor kidney m-aconitase level was altered by castration. These studies demonstrate that testosterone regulates the biosynthesis of m-aconitase in prostate epithelial cells. It appears that this is a cell-specific effect since neither kidney nor liver m-aconitase was affected. The studies reveal that prostate epithelial cells contain a constitutive and an androgen-induced m-aconitase component; whereas, kidney and liver contain a constitutive but no androgen-induced m-aconitase. Unlike essentially all other cells, m-aconitase is a regulatory and regulated enzyme in prostate epithelial cells.