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Adrenal gland reportedly expresses many nuclear receptors that are known to heterodimerize with retinoid-X-receptor (RXR) for functions, but the information regarding the glandular RXR is not adequate. Studies of rat adrenal homogenate by Western blotting revealed three RXR proteins: RXRα (55 kDa), RXRβ (47 kDa) and RXR (56 kDa). RXRγ was not detectable. After fractionation, RXRα was almost exclusively localized in the nuclear fraction. In comparison, substantial portions of RXRβ and RXR were found in both nuclear and post-nuclear particle fractions, suggesting genomic and non-genomic functions. Cells immunostained for RXRα were primarily localized in zona fasciculata (ZF) and medulla, although some stained cells were found in zona glomerulosa (ZG) and zona reticularis (ZR). In contrast, cells immunostained for RXRβ were concentrated principally in ZG, although some stained cells were seen in ZR, ZF, and medulla (in descending order, qualitatively). Analysis of adrenal lipid extracts by LC/MS did not detect 9-cis-retinoic acid (a potent RXR-ligand) but identified all-trans retinoic acid. Since C20 and C22 polyunsaturated fatty acids (PUFAs) can also activate RXR, subcellular availabilities of unesterified fatty acids were investigated by GC/MS. As results, arachidonic acid (C20:4), adrenic acid (C22:4), docosapentaenoic acid (C22:5), and cervonic acid (C22:6) were detected in the lipids extracted from each subcellular fraction. Thus, the RXR-agonizing PUFAs are available in all the main subcellular compartments considerably. The present findings not only shed light on the adrenal network of RXRs but also provide baseline information for further investigations of RXR heterodimers in the regulation of adrenal steroidogenesis.

Summary Purpose This study investigated effects of choline treatment on hepatic reticuloendothelial and biliary functions and plasma fatty acid composition in diabetic rats. Methods Diabetes was induced by streptozotocin (STZ). Choline was administered to untreated rats and a portion of STZ-treated rats for two sequences of five consecutive days, separated by a 2-day interval. Hepatic functions were studied using 99mTc Tin (II) colloid (TIN) and 99 mTc mebrofenin [bromo-iminodiacetic acid (BrIDA)] imaging. The TIN-uptake ratios (organ/whole body) of heart, liver and spleen, and the BrIDA-uptake ratios (organ or tissue/whole body) of liver, biliary tree and abdomen were obtained following imaging studies. Fatty acids were analysed by GC/MS. Results Choline treatment did not attenuate hyperglycaemic development. Diabetic rats showed (i) a decreased TIN-uptake ratio in liver with co-increased ratios in heart and spleen; choline treatment diminished these changes, (ii) elevated BrIDA-uptake ratios in biliary tree and abdomen but not in liver; choline treatment did not attenuate the elevations and (iii) decreases in plasma palmitoleic acid and oleic acid, reflecting an impaired stearoyl-CoA desaturase function; choline treatment did not affect the diminutions, but caused a decrease in arachidonic acid with a co-increase in linoleic acid. Some rats developed hypoproteinemia (HPO). HPO rats also exhibited decreases in plasma palmitoleic acid and oleic acid. Diabetes caused almost absence of palmitoleic acid in HPO rats. Choline treatment exerted no effect on the plasma fatty acid composition of diabetic HPO rats. Conclusions Choline treatment affected hepatic reticuloendothelial function and plasma fatty acid composition, but not hepatobiliary function, in diabetic rats. Whether choline treatment is beneficial requires further studies.

Prolonged stimulation with adrenocorticotropic hormone (ACTH) causes hypertension and increases Na+ intake and urine output in humans and animals. However, its biochemical basis remains to be established. Since renal Na+/H+ exchanger isoforms (NHE) and the sodium pump play an important role in this condition, their levels were examined in rats stimulated with ACTH. Male Wistar rats received daily sc injection of ACTH (30 µg/100 g of body wt) for 4 d. Half of the ACTH-stressed rats were kept for four additional days without injection of ACTH (poststimulation). In a third group, the animals were treated with dexamethasone (50 µg/100 g of body wt) daily for 4 d. A fourth group consisted of unstressed control animals. Levels of NHE proteins were measured by Western blot analysis. Sodium pump activity was assessed by the level of ouabain-sensitive K-stimulated p-nitrophenylphosphatase activity (PNP) in the renal cortex. ACTH caused a selective decrease in NHE-3, but not of NHE-1 or α-actin levels. Interestingly, this ACTH-induced change was not duplicated in the animals treated with dexamethasone. Immunofluorescence data demonstrated that NHE-3 is located in the renal proximal tubules. PNP activity, on the contrary, was increased in both the ACTH-stimulated and dexamethasone-treated animals. More important, these changes in NHE-3 and PNP activity returned to the control level poststimulation. In conclusion, while PNP upregulation may be mediated by adrenocortical glucocorticoid, a role for glucocorticoids in the suppression of NHE-3 is less clear. These changes might impair renal tubular Na+ reabsorption and hence increase Na+ and water excretion in ACTH stimulation, thus acting as a counterbalance to normalize blood pressure in ACTH-stimulated animals.

Subcellular deposition of lipofuscin granules is a marker of aging. Human and rodent adrenal cortices accumulate lipofuscin granules with age, but the mechanism that leads to the accumulation is not known. The ultrastructural appearance of lipofuscin granules resembles that of secondary lysosomes. Since adrenocortical subcellular events are predominantly influenced by ACTH action, we therefore studied the effect of prolonged ACTH-stimulation on adrenocortical accumulation of secondary lysosome-like granules, designated herein as lipofuscin granules. Using aged Fischer 344 male rats as a model, we found that a 7 day ACTH stimulation exerts a reducing effect on adrenocortical lipofuscin accumulation. Thus, adrenocortical accumulation of lipofuscin granules with age in vivo may not be an irreversible process.

Changes in the morphology of rat adrenal cortex with age include increased accumulations of lipid droplets and lipofuscin granules. Because glandular concentrations of cholesteryl esters (CE) and apolipoprotein (apo) E are also increased in parallel, the utilization or metabolism of lipid-droplet stored CE for steroidogenesis might be altered in aging cells. To explore this possibility, adrenocortical cholesterol storage and utilization were studied in 3–6 months-old (mo) (Y) rats and 20–23 mo (O) Fischer 344 male rats. Both groups received either adrenocorticotropin (ACTH1-39, Acthar gel) or gelatin alone daily for seven consecutive days. We found that: (a) the CE concentration in O rats, but not Y animals, was diminished by ACTH. The depleted CE in stimulated-O rats was replenished within five days post stimulation. Failure to deplete CE in stimulated-Y rats was not associated with an insufficient dose of the hormone, since stimulation of Y animals with higher doses of ACTH actually increased the CE concentration. In contrast, adrenocortical free cholesterol concentration remained constant during stimulation regardless of age. (b) The depleted CE in stimulated-O rats was principally comprised of cholesteryl adrenate, cholesteryl arachidonate and cholesteryl cervonate. The accumulated CE in stimulated-Y animals was primarily comprised of cholesteryl adrenate, cholesteryl arachidonate and cholesteryl oleate. (c) Whereas in stimulated-Y rats adrenal apoE concentration declined, the concentration in stimulated O animals was well maintained. (d) In vitro , adrenal homogenate or cytosolic fraction from stimulated-O rats displayed a higher capacity to hydrolyze exogenous CE than its Y counterpart. However, cholesterol esterification with external fatty acid substrates in adrenal homogenate or microsomal fraction was comparable in the two age-groups. Our findings revealed altered adrenocortical cholesterol reserve in O rats to cope with prolonged ACTH-stimulation. Changes in apoE levels and CE hydrolysis activity may be factors associated with this alteration. Depletion and accumulation of adrenocortical CE are reflected in parallel changes in cholesteryl adrenate and cholesteryl arachidonate, suggesting physiologic importance of these polyunsaturated fatty acids during sustained steroidogenesis.

A reversed phase high performance liquid chromatographic (HPLC) method was developed for direct profiling and determination of adrenal cholesteryl ester composition. Cholesteryl adrenate and cholesteryl cervonate, which are not commercially available, were synthesized as markers. Lipid extracts of rat adrenal homogenates or lipid droplets were individually applied to a conditioned silica gel-60 column which separated cholesteryl esters from other native lipids. The eluted cholesteryl ester fraction was then analyzed by HPLC. With cholesteryl heptadecanoate as internal standard, seven adrenal cholesteryl esters were detected and quantified: cholesteryl cervonate, cholesteryl arachidonate, cholesteryl adrenate, cholesteryl myristate, cholesteryl oleate, cholesteryl palmitate, and cholesteryl stearate. Among them, cholesteryl adrenate appeared to be the major sterol ester stored in the rat adrenal.

Adrenal cells from 2-6-month-old young rats (Y cells) and from 19-25-month-old aged male rats (O cells) were adapted to primary monolayer culture. The cultures of Y and O cells appeared to be primarily epithelial and rounded up in response to stimulation with adrenocorticotropic hormone (ACTH). The general morphology of O cells was comparable to that observed in Y cells except for the presence of lipofuscin-like granules, a cellular marker of aging, in O cells, but not in Y cells. ACTH-stimulated steroid production by O cells was 52% lower than that by Y cells. Exposure of intact young rats to hypoxia (0.5 atmosphere) for 21 days prior to sacrifice and culture resulted in a 122% increase of ACTH-stimulated adrenal steroidogenic activity in the cultured cells, but this effect was not observed in adrenal cells cultured from hypoxic aged rats. The results suggest that there is an age-related diminution in rat adrenal steroidogenic capacity in response to ACTH stimulation in culture derived from Y and O animals; hypoxic stress magnifies this difference.

We have previously reported that treatment of cultured mouse adrenal tumor cells with 0.6-1.2 μ M monensin, a monovalent carboxylic ionophore, results in disruption of the organized structure of the Golgi complex. This is associated with an inhibition of adrenocorticotropic hormone (ACTH) or dibutyryl cAMP-stimulated steroidogenesis and impairment of mitochondrial cholesterol side-chain cleavage activity. The present report describes further investigations regarding possible mechanisms for the inhibition. Monensin inhibits both synthesis of fluorogenic steroids and incorporation of [ 14 C]acetate into the end-product steroid 11β,20α-dihydroxy-4-pregnen-3-one. Supplementation of monensin-treated cells with 25-hydroxycholesterol, a readily available substrate for steroidogenesis, does not reverse the inhibitory effect on the reaction. The incorporation of l -[ 35 S]methionine into trichloroacetic acid precipitable proteins in the isolated mitochondria of monensin-treated cells is inhibited approximately by 40%, whereas the inhibitory effect on the proteins in the cell homogenate is marginal. These findings suggest that a deficiency of newly synthesized proteins in mitochondria, rather than the availability of the substrate cholesterol, may be the primary factor causing impairment of steroidogenesis.

To follow up an investigation which studied effects of antenatal dexamethasone therapy on neonatal respiratory performance in multifetal gestations, neonatal serum steroids were determined by HPLC. A major peak (X) whose retention time coincided with that of dexamethasone was observed in many, but not all, serum samples. However, there was no correlation between the neonates whose serum samples displayed this X-peak and the mothers who had actually received the steroid therapy, indicating that the X-substance was not dexamethasone. An alternate mobile phase was employed which separated the X-substance and dexamethasone validating the indication. Among ten clinical conditions of the neonate birth, the X-substance was found to correlate only with the mothers who had the cesarean operation for delivery, suggesting that the substance was not necessarily a steroid. Four anesthetic agents used for cesarean operations were studied; the X-substance was identified as thiopental using a LC/MS technique. This was based on the same retention times, the same negative ions at m/z 240.9 and the same daughter ions at m/z 100.8 between the two substances. Thus, caution must be exercised when HPLC is employed to study serum steroids of patients who have previously been exposed to thiopental. Moreover, recent reports have shown that thiopental affects certain metabolic reactions in the rat; the present findings also suggest a need for further investigations of thiopental effect on neonates.

Since the plasma concentration of selenium in stimulated rats was elevated, the diminution of selenium in the kidney does not seem to be secondary to a deficiency of the trace element in the circulation. In addition, the hormonal effects on both plasma and kidney selenium concentrations are likely to be mediated by adrenal glucocorticoids, because treatment of rats with dexamethasone caused similar effects. Since plasma glutathione peroxidase is primarily derived from renal proximal tubes (Avissar et al., 1994), it is conceivable that during long term stimulation of ACTH, kidney could produce and release more glutathione peroxidase into circulation by consumption of selenium in the tissue. This could provide an explanation for the findings that the enzyme activity in the kidney of stimulated rats remained unaltered, while the tissue concentration of selenium diminished.

The adrenal cortexes of humans and rodents accumulate lipofuscin with age, but the chemical nature of the substance that produces lipofuscin fluorescence in the gland is not known. Analysis of rat adrenal nonpolar lipids revealed a fluorescence profile with increased intensity in the lipids extracted from older animals (23-24 months > 6 months > 6 weeks). The peak occurred at a wavelength of 470 +/- 5 nm(n = 26) when excited at 340 nm. After sucrose density gradient centrifugation, the fluorescent substance was primarily concentrated in subcellular lipid droplets rather than supernatant or particulate. Prolonged stimulation of rats with ACTH for 7 consecutive days caused 14-51% decreases in the fluorescence levels, with a tendency of return to control levels poststimulation regardless of age. In contrast, the nonpolar lipids of mouse adrenal tumor (Y1) cells, which contain no lipofuscin, did not display this fluorescence in the presence or absence of ACTH. The chromatographic characteristics of the substance in a silica gel-60 column resembled those of authentic retinyl palmitate and cholesteryl oleate. Analysis of the substance by HPLC demonstrated at least three prominent peaks, designated XI-3 in order. X1 and X2 were minor peaks; X3 was the major peak. Whereas none of the peaks comigrated with cholesteryl esters, X1 comigrated with authentic retinyl palmitate. Determination of the fatty acid component of the major fluorescent substance X3 by gas-liquid chromatography disclosed stearic acid. Retinyl stearate was, therefore, synthesized. The fluorescent profiles, HPLC retention time and mass spectrometric fragmentation of purified X3 substance were all identical to those of the synthetic compound. In contrast, the rat liver principally accumulated retinyl palmitate with age. Thus, we conclude that 1) the major nonpolar fluorescent substance accumulated in the rat adrenal with age is retinyl stearate, which may be a fluorophore of adrenal lipofuscin; 2) ACTH action may be related to this accumulation; and 3) the type of retinyl ester accumulated in aged animals is organ specific.

Extrahepatic tissues including the adrenal cortex are capable of synthesizing apolipoprotein E (apoE). Locally synthesized apoE is believed to affect cellular uptake, transport and redistribution of cholesterol within that organ. We and another laboratory have previously reported that the adrenal cortex of aged rats has an elevated cholesteryl ester content. The aim of this work was to investigate whether this elevation is accompanied by increased adrenal apoE levels in aged rats. A Western blotting technique with polyclonal goat anti-human-apoE antiserum was employed as a probe for studies. The results showed that: (a) anti-human-apoE antiserum not only detected apoE in human plasma and adrenal homogenate, but also cross-reacted with a protein (or proteins) resembling apoE in rat plasma and adrenal homogenate (or supernatant) with a molecular weight of 34,000-36,000; (b) rat adrenal apoE concentration (per unit weight of protein) increased with age; (c) the increase did not result from blood trapped in the gland, because after organ perfusion the adrenal of aged rats persistently exhibited 58% more apoE than that of young animals. In conclusion, rat adrenal apoE concentration appears to increase with age. Whether this increase mechanistically causes the accumulation of cholesteryl esters in the aged rat adrenal remains to be investigated.

1. It is known that, in rats, hypoxia stimulates adrenal steroidogenesis, but our understanding of the hypoxic effect on the glandular parameters remains incomplete. 2. Adrenals were collected and analysed from rats that had been exposed to hypoxic conditions for 3 weeks. 3. The results reveal increased adrenal concentrations of corticosterone, free cholesterol and total cholesterol without a change in glandular weight and protein concentration. The increased total cholesterol is primarily associated with enriched cholesteryl adrenate (CE22: 4), cholesteryl arachidonate (CE20: 4) and cholesteryl oleate (CE18: 1).

responsive cells have provided useful model systems not only for the study of biochemical events associated with hormonal action, but for investigations of the possible effects of peptide hormones and other agonists on cell ultrastructure. Our laboratory has had a long standing experience with monolayer cultures of a transplantable mouse adrenocortical tumor cell line (2). We have extensively described the ultrastructural characteristics of the cells in culture and compared them with cells from the tumor of origin in vivu and normal mouse adrenocortical cells (3). In a subsequent publication we examined the effects of stimulation with ACTH by conventional transmission, high voltage and scanning electron microscopy (4). The acute steroidogenic response to ACTH is associated with a characteristic "rounding-up" of these cells. The shape changes are

The pH-dependence of fumarylacetoacetase (4-fumarylacetoacetate fumarylhydrolase, EC 3.7.1.2) activity was studied in the pH range 6.25–8.50. After correction of the substrate concentration for enolate formation, the Michaelis constant was found to be pH independent in this range. Likewise, the K i values for the competitive inhibitors chloride and fluoride were found to be independent of pH between 6.25–8.50. A bell-shaped curve described the log V vs. pH dependence, and ionization constants of 6.5 and 8.2 were calculated. Tentatively an imidazole group and a sulfhydryl group were assigned to the constants 6.5 and 8.2, respectively. Both p -hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid) react with two sulfhydryl groups per subunit in the native protein and three sulfhydryl groups per subunit in the denatured protein. Substrate protects one sulfhydryl group in the native protein from reaction with 5,5′-dithiobis(2-nitrobenzoic acid). Substrate or the comeptitive inhibitor, fluoride, protect the enzyme from inactivation by p -hydroxymercuribenzoate. In addition p -hydroxymercuribenzoate shows saturation kinetics. Neither sulfhydryl inhibitor completely inactivates the enzyme. The enzyme is described as having three sulfhydryl groups per subunit, one of which is inaccessible to the sulfhydryl specific reagents when the protein is in the native state. One of the two accessible sulfhydryl groups is either near the active site or essential in maintaining the structure of the protein.

We have developed a method for analysis of steroids in Y-1 cell cultures using reversed phase high-performance liquid chromatography (HPLC) which permits simultaneous separation and determination not only on the end-product steroid but also of other 3-keto steroid intermediates

Following 15-30 min exposure to monensin, adrenocorticotropic hormone (ACTH)- stimulated steroidogenesis in cultured adrenal cells is inhibited by 37-48 %. Electron microscopic studies reveal that, in monensin-treated cells, the Golgi complexes are disrupted into large vacuolar structures with loss of its organized structure indicating that the action of monensin on the organelles is comparably rapid. The inhibition is fully reversed after removal of the monensin-containing medium and exposure to fresh growth medium for a subsequent 4-24 h prior to stimulation. Concomitant with the restoration of full steroidogenic activity, the disrupted organelles are extensively reorganized in the cells after exposure to fresh growth medium for 4-24 h. These findings, which demonstrate, for the first time, a correlation between the morphology of the Golgi complex and steroidogenic activity, strengthen the possibility that the organelle may be involved in the regulation of steroidogenesis.

It has previously been shown that the steroidogenic action of adrenocorticotropic hormone (ACTH) is accompanied by characteristic alterations in cell ultrastructure. These include hypertrophy of the Golgi complex associated with increased vesicle formation and striking elevations of acid phosphatase activity in the Golgi complex and lysosomes. To investigate a possible relationship of these phenomena to steroidogenic function in monolayer cultures of murine adrenal tumor cells, monensin, a carboxylic ionophore which disrupts the ordered structure and transport function of the Golgi complex, was used. Monensin, at a concentration of 1.2 microM, causes massive vacuolization and hypertrophy of the Golgi complex. No effect on mitochondrial structure was seen. Monensin, 0.6-1.2 microM, inhibits both ACTH-stimulated and basal steroidogenesis by approximately 50% in incubations of 4 h or less. Dibutyryl-cAMP-stimulated steroidogenesis was inhibited to a similar degree. Incubations were carried out in serum-free media to eliminate possible effects due to exogenous cholesterol transport into the cell. There were no direct inhibitory effects of monensin on cholesterol side-chain cleavage (SCC) activity in isolated mitochondria. In contrast, mitochondria isolated from cells previously treated with monensin had a reduced capacity for this activity. These experiments suggest that monensin inhibits transport of cholesterol from the Golgi complex to the mitochondrial site of steroidogenesis action or interferes with the transport of key mitochondrial proteins synthesized on cytoplasmic ribosomes.

Inner and outer submitochondrial membranes were prepared after disruption of malate-treated bovine adrenocortical mitochondria. It was found that a part of the endogenous cholesterol in the inner membrane (approx. 50%) was rapidly utilized by the cholesterol side-chain cleavage reaction. The utilization of cholesterol in the outer membrane, on the other hand, was inefficient and slow in spite of the fact that cholesterol concentration is higher in the outer than in the inner membrane. When the inner membrane prepared from untreated mitochondria was incubated for 20 min in the presence of a reconstituted cytochrome P-450-reducing system, the inner membrane cholesterol was depleted by approximately 70%. The half-life of the depletion reaction was 2-3 min. In addition, when the outer membrane plus the soluble fraction from the untreated mitochondria were added as a source of cholesterol to the inner membrane fraction, a marginal increase in the production of steroids was observed. From these results it is concluded that a portion of the inner membrane cholesterol can be steroidogenic, whereas the rest of the cholesterol is non-steroidogenic.

The cholesterol contents and phospholipid compositions of mitochondria, microsomes and submitochondrial membranes from bovine adrenal cortex have been analyzed quantitatively. From our results, the following cholesterol contents were obtained: mitochondria, 6.2 +/- 0.9 mol %; microsomes, 18.4 +/- 2.8 mol %; mitochondrial inner membrane, 2.8 +/- 0.6 mol %; and mitochondrial outer membrane, 8.3 +/- 1.3 mol %. In addition, the phospholipid compositions of the mitochondrial inner and outer membranes were determined for the first time. Cardiolipin was found to be enriched in the inner membrane, whereas phosphatidylinositol was richer in the outer membrane. The general features of phospholipid compositions in the submitochondrial membranes resembled that of rat liver mitochondria.