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Background: Prostate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells., Results: The studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells., Conclusion: This study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

The control and alteration of key regulatory enzymes is a determinant of the reactions and pathways of intermediary metabolism in mammalian cells. An important mechanism in the metabolic control is the hormonal regulation of the genes associated with the transcription and the biosynthesis of these key enzymes. The secretory epithelial cells of the prostate gland of humans and other animals possess a unique citrate-related metabolic pathway regulated by testosterone and prolactin. This specialized hormone-regulated metabolic activity is responsible for the major prostate function of the production and secretion of extraordinarily high levels of citrate. The key regulatory enzymes directly associated with citrate production in the prostate cells are mitochondrial aspartate aminotransferase, pyruvate dehydrogenase, and mitochondrial aconitase. Testosterone and prolactin are involved in the regulation of the corresponding genes associated with these enzymes (which we refer to as "metabolic genes"). The regulatory regions of these genes contain the necessary response elements that confer the ability of both hormones to control gene transcription. In this report, we describe the role of protein kinase c (PKC) as the signaling pathway for the prolactin regulation of the metabolic genes in prostate cells. Testosterone and prolactin regulation of these metabolic genes (which are constitutively expressed in all mammalian cells) is specific for these citrate-producing cells. We hope that this review will provide a strong basis for future studies regarding the hormonal regulation of citrate-related intermediary metabolism. Most importantly, altered citrate metabolism is a persistent distinguishing characteristic (decreased citrate production) of prostate cancer (PCa) and also (increased citrate production) of benign prostatic hyperplasia (BPH). An understanding of the role of hormonal regulation of metabolism is essential to understanding the pathogenesis of prostate disease. The relationships described for the regulation of prostate cell metabolism provides insight into the mechanisms of hormonal regulation of mammalian cells in general.

Background: 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., Methods: 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., Results: 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., Conclusions: 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., (Copyright 2000 Wiley-Liss, Inc.)

The prostate gland of humans and other animals has the unique function of accumulating and secreting extraordinarily high levels of citrate. The prostate secretory epithelial cells synthesize citrate which, due to a limiting mitochondrial (m-) aconitase, accumulates rather than being oxidized. Thus citrate is essentially an end product of metabolism in prostate. For continued net citrate production, a continual source of oxaloacetate (OAA) and acetyl CoA is required. Glucose via pyruvate oxidation provides the source of Acetyl CoA. In prostate cells, citrate production is regulated by testosterone and/or by prolactin. Both hormones selectively regulate the level and activity of pyruvate dehydrogenase E1 alpha (E1a) in animal prostate cells; thereby regulating the availability of acetyl CoA for citrate synthesis. Studies were conducted to determine if testosterone and prolactin might regulate the expression of the E1a gene in prostate epithelial cells. Prolactin treatment of rat ventral and lateral prostate cells and human PC3 cells increased the levels of E1a mRNA and the rates of transcription of the E1a gene. Testosterone also increased the mRNA level and transcription of E1a in rat ventral prostate cells, and in PC3 cells transfected with androgen receptor. However, testosterone treatment resulted in a repression of E1a gene expression in lateral prostate cells. Evidence is presented which supports the view that prolactin regulation of E1a is mediated via PKC. The rapidity of the effects of both hormones is representative of an immediate-early gene response. To our knowledge this represents the first report in any mammalian cells that, in addition to its constitutive expression in all mammalian cells, the E1a gene is a hormonally-regulated gene in specifically targeted prostate epithelial cells.

The glandular epithelial cells of the human prostate gland have the unique capability and function of accumulating the highest zinc levels of any soft tissue in the body. Zinc accumulation in the prostate is regulated by prolactin and testosterone; however, little information is available concerning the mechanisms associated with zinc accumulation and its regulation in prostate epithelial cells. In the present studies the uptake and accumulation of zinc were determined in the human malignant prostate cell lines LNCaP and PC-3. The results demonstrate that LNCaP cells and PC-3 cells possess the unique capability of accumulating high levels of zinc. Zinc accumulation in both cell types is stimulated by physiological concentrations of prolactin and testosterone. The studies reveal that these cells contain a rapid zinc uptake process indicative of a plasma membrane zinc transporter. Initial kinetic studies demonstrate that the rapid uptake of zinc is effective under physiological conditions that reflect the total and mobile zinc levels in circulation. Correspondingly, genetic studies demonstrate the expression of a ZIP family zinc uptake transporter in both LNCaP and PC-3 cells. The rapid zinc uptake transport process is stimulated by treatment of cells with physiological levels of prolactin and testosterone, which possibly is the result of the regulation of the ZIP-type zinc transporter gene. These zinc-accumulating characteristics are specific for prostate cells. The studies support the concept that these prostate cells express a unique hormone-responsive, plasma membrane-associated, rapid zinc uptake transporter gene associated with their unique ability to accumulate high zinc levels.

The prostate gland of many animals accumulates extremely high levels of zinc and citrate. Evidence currently exists in support of a concept that zinc might be an important regulator of m-aconitase and citrate oxidation of prostate epithelial cells. No information concerning the mitochondrial levels of zinc has been available. The roles of testosterone and prolactin in the regulation of zinc have not been established. In this report, we determined the levels of tissue and mitochondrial zinc of rat lateral prostate (LP), ventral prostate (VP), dorsal prostate (DP), liver, and kidney. The results demonstrate that the mitochondrial zinc levels of the prostate cells were higher than levels of nonprostatic cells. The LP contained severalfold higher zinc levels than DP and VP. The effects of testosterone and prolactin in vivo and in vitro on the zinc levels were also determined. Both hormones significantly increased cellular and mitochondrial zinc levels of LP cells; decreased the zinc levels of VP cells; and had no effect on the zinc levels of DP, liver, or kidney cells. These effects are direct and physiological effects of the hormones on the targeted prostate epithelial cells. The hormonal effects on mitochondrial zinc of LP and VP epithelial cells correlate perfectly with their effects on citrate oxidation. The results support the concept that mitochondrial zinc is an inhibitor of m-aconitase and citrate oxidation; and that prolactin and testosterone regulation of mitochondrial zinc provides a mechanism for their regulation of citrate oxidation in citrate-producing prostate epithelial cells.

Objectives: The function of the prostate gland in many animals, including humans, is to accumulate and secrete 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 of testosterone 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 microgram/mL), or vehicle (control)., Results: Both hormone applications caused a marked increase in the level of m-aconitase. In contrast, neither hormone 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 sub-sequent citrate oxidation of specific prostate epithelial cells. This unique aconitase relationship is not observed in other mammalian cells.

Mitochondria (m-)aconitase is a rate-limiting regulatory enzyme in prostate epithelial cells which minimizes citrate oxidation by these cells. This unique metabolite 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.(ABSTRACT TRUNCATED AT 250 WORDS)

A major and unique function of the prostate is the production of extremely high levels of citrate. Testosterone is an important regulator of this function. This regulation is achieved by hormonal stimulation of citrate synthesis by prostate secretory epithelial cells. Previous studies have established that testosterone stimulates the mitochondrial transamination of aspartate to OAA for citrate synthesis. However, increased citrate synthesis would also require an increased availability of AcCoA which is derived from pyruvate. The present studies were concerned with the in vivo effect of testosterone on pyruvate dehydrogenase (PDH) activity of prostate mitochondria isolated from rat ventral prostate (VP). The results demonstrated that testosterone administration to castrated rats stimulates PDH activity and citrate production from pyruvate in the presence of OAA. Therefore testosterone increases both OAA and acetyl CoA production which results in increased citrate synthesis. The mechanism of testosterone regulation of PDH can now be investigated.

The prostate gland produces and secretes extraordinarily high levels of citrate. Studies with rat ventral prostate (VP) have demonstrated that aspartate can serve as a four-carbon source of oxalacetate in the synthesis of citrate. To achieve this, prostate secretory epithelial cells must contain a transport system for the active uptake of aspartate from circulation. The present studies with VP epithelial cells confirm the existence of a Na(+)-dependent high-affinity L-aspartate transporter. The transporter has an optimal pH approximately 7.5 and is temperature dependent. It appears to be an anionic amino acid transporter capable of transporting L-glutamate but not basic or neutral amino acids. The transporter is inhibited by ATPase inhibitors, thereby indicating its dependency on a Na+ gradient. The characteristics of the high-affinity L-aspartate transporter are consistent with its operation at the basilar membrane for the transport of circulating aspartate into the cell. Castration (24 hr) resulted in a significant decrease in the ability of VP epithelial cells to transport L-aspartate. The administration of testosterone to castrated rats completely restored L-aspartate transport. In addition, in vitro testosterone addition (10(-8) M for 30 min) to isolated prostate epithelial cells markedly increased L-aspartate transport. Both cycloheximide and actinomycin inhibited the testosterone effect. The studies reveal that testosterone is a regulator of this Na(+)-dependent high-affinity L-aspartate transporter. The mechanism of this testosterone effect appears to involve both RNA and protein synthesis. We now have a model system to elucidate this novel effect of testosterone.

The effect of testosterone on the precursor mitochondrial aspartate aminotransferase (pmAAT) gene and on pmAAT-mRNA was studied in rat ventral prostate (VP) and pig prostate epithelial cells. Castration significantly decreased the level of nuclear pmAAT transcripts in VP; whereas testosterone treatment of castrated animals restored the level of pmAAT transcripts. Correspondingly, castration resulted in a marked decrease in the transcription rate of the pmAAT gene; whereas testosterone treatment markedly increased the transcription rate. In vitro studies with isolated pig prostate epithelial cells demonstrated that testosterone directly and rapidly induced a transient increase in the transcription rate of the pmAAT gene. The increase in transcription was associated with an increase in the steady-state level of pmAAT-mRNA. Similar in vitro effects were observed with isolated VP epithelial cells. In addition to its stimulatory effect on transcription of the pmAAT gene, testosterone also increased the half-life of pmAAT-mRNA from 2 h in the absence of hormone to 16 h in its presence. Consequently, testosterone appears to stabilize the pmAAT-mRNA. The combination of its immediate effect on stimulating the transcription of the pmAAT gene and its stabilizing effect on pmAAT-mRNA would account for the increase in the steady-state level of pmAAT-mRNA by testosterone. These studies support our proposal that, through these effects, testosterone increases the biosynthesis of mAAT thereby increasing the transamination of aspartate to oxaloacetate and ultimately increasing the synthesis of citrate. This appears to provide at least one of the mechanisms by which testosterone regulates prostate citrate production.

The effect of testosterone on precursor mitochondrial aspartate aminotransferase (pmAAT) mRNA was studied in rat ventral prostate and primary cell cultures of mini-pig prostate. Testosterone induced a 2-3-fold increase in pmAAT mRNA level in both rat ventral prostate and mini-pig prostate cultures. The pmAAT mRNA induction occurred 30 min after testosterone treatment and was maximal by 1.5 h. Prostatic mAAT activity was also induced by testosterone with a 1-2 h lag period. The time-course of induction of pmAAT mRNA, pmAAT activity and mAAT activity was consistent with stimulation of mRNA synthesis followed by increased synthesis and import of pmAAT into mitochondria. The effect of testosterone on pmAAT mRNA was specific because the increase in pmAAT mRNA was at least 2-fold greater than the increase in poly (A+) RNA. These results suggest that testosterone stimulated mAAT activity by induction of pmAAT mRNA. This continues to support our proposal that a major physiological effect of testosterone is increased pmAAT mRNA steady-state levels which result in increased pmAAT synthesis and increased mAAT activity. These changes ultimately result in increased citrate production by prostate epithelial cells.

Mitochondrial aspartate transamination was investigated as a major source of oxalacetate for citrate synthesis in rat ventral prostate. Citrate accumulation was measured in isolated mitochondria incubated with acetyl coenzyme A and various combinations of amino acids. Aspartate plus alpha ketoglutarate in the presence of acetyl coenzyme A resulted in significant citrate accumulation. Neither aspartate nor alpha ketoglutarate alone resulted in any significant citrate accumulation. Aspartate and alpha ketoglutarate use was comparable to glutamate and citrate production. The results indicated the presence of a mitochondrial aspartate aminotransferase. Castration (3 days) caused a significant decrease in citrate production from aspartate plus alpha ketoglutarate as well as a decrease in mitochondrial AAT activity in prostate although no effect on kidney activity occurred. A single injection of 1 mg. testosterone propionate to castrate rats significantly increased prostate mitochondrial AAT activity within 24 hours while MDH activity was unaltered. A double reciprocal plot indicated that testosterone might regulate the level of mitochondrial AAT in prostate. Ventral prostate also contain a uniquely high level of endogenous aspartate. These studies indicate that aspartate might be the major 4-carbon source of oxalacetate for citrate synthesis. Also testosterone possibly regulated prostate citrate production by its effect on the level of mitochondrial AAT activity.

Citrate oxidation by rat ventral prostate was reduced by castration and increased by testosterone administration. Similarly, the mitochondrial aconitase activity was decreased by castration; whereas cytosol aconitase was unaffected. The rate of citrate oxidation is extremely low in prostate. Castration also decreased mitochondrial aspartate aminotransferase activity while having no effect on the cytosol isoenzyme. Testosterone markedly stimulated the net production of citrate from aspartate plus glutamate by prostate mitochondria. These studies support the proposal that aspartate is a major source of oxalacetate for citrate production, and that a "glutamate-aspartate-citrate" pathway may be functional in prostate mitochondria. In addition, testosterone can regulate citrate production by a specific effect on mitochondrial aspartate aminotransferase activity. Testosterone also regulates the flux of citrate through the Krebs cycle, but this represents only a small proportion of the citrate accumulated. These conditions would be consistent with the function of prostate epithelium in accumulating and secreting citrate.

Stimulation of mitochondrial aspartate aminotransferase (mAAT) activity by testosterone was determined in organ cultures of rat ventral prostate. The effect of testosterone on citrate accumulation in the culture medium was also determined. Testosterone stimulation of citrate accumulation and mAAT occurred in a dose dependent manner. Stimulation of mAAT activity occurred after a 1-3 h lag period and appeared to involve the synthesis of specific RNA since the response was inhibited by actinomycin D. Studies utilizing [3H]L-leucine indicated that unlike the total tissue, testosterone stimulated the incorporation of [3H]leucine into proteins of the mitochondrial fraction. The results suggested that mitochondrial proteins may be more sensitive to testosterone stimulation than cytosol proteins. The response was specific for mAAT since testosterone had no effect on mitochondrial malic dehydrogenase activity. The data suggested that testosterone may regulate prostate citrate content by the induction of mAAT in prostate mitochondria, which results in a source oxalacetic acid for citrate synthesis through transamination of aspartate by alpha ketoglutarate.

The prostate gland is unusual in that it has a high content of citric acid compared with other soft tissues. The mechanism of citrate accumulation in prostate is unknown. One possibility is that aspartate aminotransferase (AAT) could supply an extra Krebs cycle source of oxaloacetate for citrate formation. Since testosterone regulates citrate production and secretion and strong transaminase activity exists in prostate, a relationship between AAT and citrate production could exist if testosterone also regulates AAT activity. To investigate this possibility, animals were castrated and given testosterone. After a total castration period of 72 hours, animals were sacrificed and mitochondrial AAT activity determined. Results revealed that castration decreased AAT activity and testosterone replacement stimulated activity. Neither castration nor testosterone affected malate dehydrogenase (MDH) activity. Both actinomycin D and cycloheximide blocked the testosterone stimulation of AAT activity. These studies indicate that testosterone may regulate AAT activity in rat ventral prostate. Furthermore, regulation may occur through testosterone stimulation of specific RNA and protein synthesis.

The effects of testosterone on mitochondrial aspartate aminotransferase (mAAT) synthesis in rat ventral prostate was investigated. Procedures for the isolation, purification and characterization of AAT isozymes were developed and described. Purified mAAT preparations contained no demonstrable contaminating proteins. Prostatic mAAT was characterized as a cationic protein with an estimated mol. wt of 120,000. Cytoplasmic AAT (cAAT) isozyme was identified as an anionic protein with an estimated mol. wt of 132,000. A cytosolic cationic isozyme, similar to mAAT, was also identified as pre-mAAT. Testosterone administration to castrated rats resulted in significant increases in leucine incorporation into mAAT, in the level of mAAT, and in mAAT activity. These effects of testosterone were observed within 2 h of administration. Conversely, testosterone administration had none of these effects on cAAT or on non-AAT protein pool. Testosterone treatment did appear to increase leucine incorporation into pre-mAAT. Testosterone treatment in organ cultures and in prostate epithelial cell cultures resulted in the same stimulatory effects on mAAT as observed in the in vivo studies. The hormone was effective at the physiological concentration of 2 X 10(-9) M. These results indicated that testosterone has a rapid and specific effect on the biosynthesis of mAAT. This continues to support our proposal that testosterone regulates prostate citrate production via a stimulatory effect on mAAT which results in increased mitochondrial synthesis of citrate from aspartate.

The effects of liposome-encapsulated diethylstilbestrol (DES) on the size of the ventral prostate gland and total testosterone levels in rats were studied. DES was encapsulated in phosphatidycholine-cholesterol-stearylamine-alpha-tocopherol containing multilamellar liposomes in a molar ratio of 4:5:1:0.01. The liposome suspension or free DES plus blank liposomes were administered sc on days 1, 3, and 5 at a dose of 400, 87, or 12 micrograms/kg. Plasma testosterone values showed a decrease with dose for animals treated with either entrapped DES or free DES. Prostate gland weight per g animal and prostatic protein mg per g animal on day 7 were significantly decreased in the treated animals compared to those in controls dosed with DES plus buffer containing liposomes. In addition, there was a body weight loss for the high dose DES-encapsulated animals and for high and medium dose DES plus blank liposome-treated and blank liposome-treated animals over the treatment period, although only the DES-encapsulated group value was statistically significant (P less than 0.05). Animals at the other doses did not lose weight, but exhibited weight gains which were less than those observed for sham-injected animals. Rats that received 7 micrograms/kg liposome-entrapped DES for 4 weeks had significantly lower prostate gland weights, prostatic protein levels, and testosterone levels than control animals receiving the same regimen of free DES plus blank liposomes. Results from the studies indicate that liposome-entrapped DES was more effective in decreasing the size of the prostate gland and that the desired therapeutic end point (prostate ablation) may be attained with a lower dose of DES, which would decrease the risk of weight loss in the experimental animals.