Biomedical subjects
Publications and source records attributed to S Azhar.
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The activities of three enzymes--two mitochondrial and one microsomal--were measured in isolated islets of Langerhans from 2-month-old and 12-month-old rats. Mitochondrial glycerophosphate dehydrogenase activity (expressed as nanomoles of iodonitrotetrazolium reduced per minute per milligram of protein), decreased (P less than 0.01) from a mean (+/- SEM) of 73.2 +/- 11.2 (2-month-old) to 34.7 +/- 5.9 (12-month-old). In contrast, activities of neither mitochondrial monoamine oxidase nor microsomal NADH cytochrome-c reductase changed with age. These results demonstrate that the activity of the glycerophosphate shuttle decreases as rats grow older, and it raises the possibility that the consequent difficulty in regenerating cytosolic NAD+ may play a role in the insulin secretory defect associated with aging.
Luteal cells of immature female rats treated with gonadotropins contain microtubules with a number of interesting features. Many of the microtubules of these cells are arranged in bundles in which they are separated one from another by strands of material (i-MT bands) of unknown composition. The microtubules within the bundles assume a hexagonal packing pattern with i-MT bands between any two microtubules. The bundle microtubules and their i-MT bands are further connected via crosslinking filaments: pattern obtained from densitometer scans (measuring the arrangement of the crosslinking filaments) suggest that the filaments may represent microtubule-associated proteins. The complex arrangement of the microtubules within a bundle does not appear to extend for the entire length of the individual microtubules, and occasionally one sees profiles of single microtubules fanning out from the ends of the bundle: whether the same microtubules are regrouped at some other point in the cell is not known. Structures similar to the i-MT band and the crosslinking filaments have also been observed connecting microtubules to segments of the luteal cell plasma membrane: in these instances the i-MT-like band is found between the longitudinally sectioned microtubule and the membrane, with filaments connecting the two structures via the intermediate band. It is of interest that the microtubules of these luteal cells are not sensitive to treatment with antimicrotubule drugs and we suggest that the complex bundling arrangement provides their unusual stability.
In rats, muscle glycogen depletion has been associated with increased insulin action. Whether this also occurs in man has not been reported. After 4 d rest, 13 males (E Group) had a percutaneous muscle biopsy of the vastus lateralis muscle followed by a euglycemic clamp at plasma insulin congruent to 100 microU/ml and congruent to 1,900 microU/ml, with simultaneous indirect calorimetry. This was repeated 1 wk later, but after glycogen-depleting exercise the night before the euglycemic clamp. Seven subjects underwent the same protocol but were also re-fed 100 g carbohydrate (CHO) after the exercise (EF group). In both groups, the mean muscle glycogen content was approximately 40% lower (P less than 0.01) after exercise compared with the muscle glycogen content measured after rest. In the E group, the mean muscle glycogen synthase activity (percent independent of glucose-6-phosphate) increased threefold (P less than 0.001) after exercise, but increased only twofold in the EF group (P less than 0.02 between groups). In both groups, the mean basal and insulin-stimulated CHO oxidation rates were lower in the post-exercise, glycogen-depleted condition compared with the rested, glycogen-replete condition. The mean insulin-stimulated CHO storage rate increased significantly in the E group after exercise but not in the EF group. In the E group, the total insulin-stimulated CHO disposal rate (M) was 17 (P less than 0.04) and 10% (P less than 0.03) higher after exercise during the low and high dose insulin infusion, respectively. No significant changes in M were observed in the EF group. For all subjects, after rest and exercise, the M correlated with the CHO storage rates during the low (r = 0.80, P less than 0.001) and high dose (r = 0.77, P less than 0.001) insulin infusions. After exercise, the muscle glycogen synthase activity correlated with the CHO storage rate (r = 0.73, P less than 0.002; r = 0.75, P less than 0.002) during the low and high dose insulin infusions, respectively, and also with M (r = 0.64, P less than 0.008; r = 0.57; P less than 0.02).
Rat steroidogenic tissues take up cholesterol, and it has been suggested that this process plays a regulatory role in steroid hormone synthesis. To provide evidence for this hypothesis, we carried out studies in lipoprotein-deficient rats. Lipoprotein deficiency, achieved by treating male rats with pharmacological amounts of estradiol, led to profound lowering of plasma cholesterol (8 +/- 2 versus 54 +/- 4 mg/dl) and adrenal cholesteryl ester content (113 +/- 57 versus 747 +/- 108 micrograms/organ). Basal serum corticosterone levels were decreased by 50%, and the response to adrenocorticotropic hormone (ACTH) was totally abolished. Injection of high density lipoprotein (HDL) to estradiol-treated animals restored the response of corticosterone to ACTH. Comparable in vitro studies with adrenal cell suspensions obtained from lipoprotein-deficient rats confirmed the in vivo data. Measurement of [14C]acetate incorporation and uptake of both HDL- and low density lipoprotein (LDL)-cholesterol in these adrenal cells showed a progressive increase with the duration of estradiol treatment, and neither of these two phenomena was altered by ACTH. These results provide in vitro and in vivo evidence for the hypothesis that normal adrenal steroidogenesis depends upon cholesterol delivery from plasma. Furthermore, under the conditions studied, ACTH does not stimulate adrenal de novo cholesterol biosynthesis nor the uptake of either HDL- or LDL-cholesterol.
The present studies were performed to evaluate the role of steroid precursors and plasma lipoproteins in gonadal tissue steroidogenesis. Leydig cell suspension isolated from rat testes responded to hCG. Bt2cAMP, 8 Br-cAMP and cholera toxin with an increase in testosterone response. Administration of 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) reduced the plasma cholesterol and testosterone levels in a time and dose dependent manner. This treatment also reduced the steroidogenic capacity of isolated Leydig cells both under basal conditions and in response to trophic hormone. Different doses of 4-APP up to 25 mg/kg BW and up to 4 days of treatment, however, did not modulate cholesterol and cholesterol ester contents of isolated Leydig cells. 4-APP treatment also had no effect on testis weight, phospholipid content, protein synthesis and energy metabolism in isolated Leydig cells. Similarly, administration of 4-APP (12.5 mg/kg) to PMSG-hCG primed rats beginning on day 3, post hCG, drastically reduced the circulating cholesterol and progesterone levels. Injection of the drug also produced an inhibition in vitro luteal cell steroidogenesis and a reduction in cellular cholesterol esters and free cholesterol contents. Addition of LDL or HDL to incubation medium reversed the inhibitory effect of 4-APP on luteal cell steroidogenesis while this inhibition persisted in Leydig cells. Injection of rats with Triton-WR-1339 (mg/kg BW) resulted in a 10-fold increase in plasma cholesterol and a contrasting decrease in testosterone levels. This treatment, however, produced no effect on in vitro Leydig cell steroidogenesis or cellular content of cholesterol esters and free cholesterol. It appears that the Leydig and luteal cells process and utilize lipoprotein-delivered cholesterol for steroidogenesis through different mechanism(s). These studies thus demonstrate differential actions and an acute regulatory role of lipoproteins in gonadotropin modulated steroidogenesis in two different gonadal tissue.
Islest from different regions of pancreases of aging rats were compared for size and variations of response to glucose stimulation. The results show that pancreatic islets from the ventral-duodenal and splenic regions of 12-mo-old retired breeder. Spraque dawley rats are comparable in all respects measured: thus, pancreatic regional differences cannot explain the age-associated reduction in beta cell secretory response noted in previous studies.
This report represents an effort to reinvestigate the relationship of steroid hormone processing with microtubule protein, using the highly active progesterone-producing cells of superovulated immature rats as a model steroid hormone system. For the most part, gonadotropin-primed rats were used at 6 days and were reinjected with saline or various doses of colchicine and vinblastine: after 3 h the animals were injected with human chorionic gonadotropin (hCG, 10 IU) or saline for an additional hour. Subsequently, the ovaries were perfused with fixative for morphological studies or luteal cells were isolated for in vitro incubation with various additives. The results are as follows: 1) luteal cell progesterone synthesis is reduced in a dose- and time-dependent manner after treatment with antimicrotubule drugs; 2) additional stimulating agents given in vitro (hCG, Bt2cAMP, epinephrine) do not overcome the reduction produced by treatment with antimicrotubule drugs; 3) luteal cells show normal protein synthesis after treatment with antimicrotubule drugs; 4) luteal cells have microtubules of unusual appearance that are insensitive to the action of antimicrotubule agents. These results show that in the luteinized ovary, both colchicine and vinblastine interfere with hormone-stimulated steroidogenesis. Although the mechanism for the effect is not yet understood, it does not appear to be related to the content of the intact microtubules in luteal cells. Whether the drugs exert their action by binding to the unassembled form of microtubule protein in the cells remains to be determined.
Ketoconazole, a new oral drug used to treat systemic and superficial mycoses, inhibits sterol synthesis in fungi. The development of gynecomastia in two patients prompted us to investigate the effect of the drug on testosterone production. After a 200-, 400-, or 600-mg dose, volunteer male testosterone serum concentrations fell markedly, but returned toward baseline eight to 24 hours later as ketoconazole serum concentrations waned. A marked but transient drop in testosterone levels occurred in patients receiving long-term therapy, and continuous testosterone depression was noted in one. A block of synthesis was demonstrated in vitro. Ketoconazole at concentrations achievable in serum with currently used doses blocked basal and gonadotropin-stimulated testosterone production by rat Leydig cells. The diminution of testosterone synthesis could be significant as further therapeutic trials may use larger doses or more than once-daily administration. The paucity of reports of endocrinologic toxicity may relate to the "escape" from the block demonstrated in vivo.
Pretreatment of ovarian cells with concanavalin A and wheat-germ agglutinin blocked the gonadotropin-induced cyclic AMP and progesterone responses and this effect was time- and concentration-dependent. Basal production of either cyclic AMP or progesterone, however, was not affected by treatment of cells with lectin. The effect of concanavalin A on gonadotropin-mediated cyclic AMP and progesterone responses was blocked by alpha-methyl D-mannoside and alpha-methyl d-glucoside. Similarly the inhibitory effect of wheat-germ agglutinin was reversed by N-acetyl-D-glucosamine. Pretreatment of ovarian cells with concanavalin A or wheat-germ agglutinin had no effect on protein synthesis in the ovary as monitored by [3H]proline incorporation studies. Concanavalin A and wheat-germ agglutinin did not affect steroid production in response to dibutyryl cyclic AMP and 8-bromo cyclic AMP, indicating that the inhibitory action of lectin was occurring at a step before cyclic AMP formation. Lectins specific for L-fucose, D-galactose and N-acetyl-D-galactosamine, gorse seed agglutinin, peanut agglutinin and Dolichos biflorus agglutinin respectively, did not interfere with gonadotropin-induced cyclic AMP and progesterone responses. The present studies suggest that gonadotropin receptors may be glycoprotein in nature or closely associated with glycoprotein structures with the carbohydrate chain containing N-acetyl-D-glucosamine, mannose and possibly N-acetylneuraminic acid.
Incubation of luteal cells with human, horse and rat sera, but not bovine sera resulted in enhanced basal and hCG-stimulated progesterone accumulation. The stimulatory effect of human or rat sera on basal, hCG- or 8 Br-cyclic AMP-induced progesterone synthesis in luteal cells was evident within 15-30 min after incubation, reaching a maximum after 3-4 h. The stimulatory effects of hCG and/or sera were blocked by inhibitors of RNA and protein synthesis. Similarly, lysosomotropic agents, chloroquine (100 microM) and ammonium chloride (10 mM), partly blocked the steroidogenic response of luteal cells to hCG and/or human or rat sera. Incubation of cells in the presence of 2-deoxyglucose, sodium azide and phenylmethylsulfonyl fluoride resulted in partial inhibition of progesterone secretion in response to hCG or sera. Fractionation of human or rat sera into various lipoprotein fractions demonstrated that LDL and HDL most effectively supported and potentiated the steroidogenic response to hCG. Lipoprotein-deficient serum, however, did not alter gonadotropin-induced steroid production. Incubation of luteal cells with increasing concentrations of h-LDL and h-HDL enhanced both basal and hCG-mediated steroidogenesis in a dose-related manner, although very high concentrations of these lipoproteins were inhibitory. Further, [3H]cholesterol from [3H]cholesteryl linoleate-LDL was incorporated into luteal cell progesterone and the extent of this incorporation was enhanced by hCG. Addition of excess unlabeled h-LDL, h-HDL, as well as r-HDL, drastically reduced the incorporation of radioactive label into progesterone. These studies suggest that (a) serum potentiation of steroidogenesis was due to presence of lipoproteins, mainly LDL and HDL, and (b) the lipoprotein-bound cholesterol is delivered into the luteal cells and utilized for steroidogenesis.
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The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.
This report describes an interaction between rat brain microtubule protein and various hepatic fractions in vitro. Purified preparations of Golgi membranes, plasma membrane, rough and smooth endoplasmic reticulum, nuclear membranes, and mitochondria were obtained from the livers of 200-g rats. Several concentrations of fresh or sonicated frozen membranes were incubated with twice-cycled rat brain microtubule protein in a microtubule assembly buffer for 60 min at 30 degrees C. Changes in microtubule assembly were assessed either by quantitative electron microscopy on negatively stained samples or by spectrophotometric methods. The results show that all the tested membranes "bound" microtubule protein, preventing assembly: Golgi and plasma membranes, as well as mitochondria, were especially potent in this regard. To identify the membrane-associated components responsible for microtubule protein binding, the membranes were extracted with methanol-chloroform, and liposomes were prepared from the resulting lipids. Microtubule protein incubated with these liposomes showed a differential ability to assemble that was similar to the effect obtained with intact membranes. Membrane-extracted phospholipids were identified as the lipid component responsible for these changes, with the negatively charged phospholipids (cardiolipin and phosphatidylserine) being uniquely active. These findings indicate that hepatic membranes differentially interact with brain microtubule protein; this interaction may be dependent on membrane phospholipids.
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The action of danazol on 125I-human chorionic gonadotropin (hCG) binding, gonadotropin-stimulated adenosine 3',5' cyclic monophosphate (cAMP) accumulation and progesterone production has been investigated in luteinized rat ovaries. Preincubation of luteal cells for short periods of time with increasing concentrations of danazol caused a significant inhibition of gonadotropin-stimulated steroidogenesis. The inhibitory effect of danazol was both concentration and time dependent. Danazol also reduced progesterone production in response to cholera enterotoxin and 8 bromo-adenosine-cAMP, but it had no effect on hCG, luteinizing hormone, and cholera enterotoxin stimulated cAMP formation. Similarly danazol did not affect 125I-hCG binding as assessed by the equilibrium dissociation constant (Kd) and number of hormone-binding sites on the luteal cell surface. These results suggest that in intact luteal cells danazol inhibits steroidogenesis at a point distal to hormone-receptor interaction and cAMP formation.
The administration to female rats of the LHRH agonist, [des-GlyNH2(10)]LHRH ethylamide, as either a single dose or repeated injections resulted in a decrease in the binding of 125I-labeled hCG to ovarian plasma membranes. An ovarian cell suspension prepared from the agonist-injected group of rats also responded to hCG with a decreased ability to accumulate cAMP and progesterone compared with the saline-treated controls. The receptor loss was due to a decreased number of hormone-binding sites rather than to a change in the affinity of the receptor for the hormone. Preincubation of ovarian cells with the agonistic analog in vitro also caused an inhibition of progesterone synthesis in response to hCG, cholera toxin, and 8-Bromo-cAMP, suggesting a direct inhibitory effect of the analog on ovarian steroidogenesis. Prior exposure to the agonistic analog, however, had no effect on cAMP accumulation by the ovarian cells in response to either hCG or cholera enterotoxin, further suggesting that the site of inhibitory action lies at a point after cAMP accumulation. In summary, the present study demonstrates that 1) the inhibition of ovarian steroidogenesis observed in response to the injection of the LHRH agonist is primarily due to down-regulation of ovarian hCG/LH receptors with a resultant decrease in cAMP accumulation, and this effect may be caused by the pulses of LH release from the pituitary, and 2) the direct inhibitory effect on steroidogenesis observed under in vitro conditions is independent of gonadotropin receptor, and the site of this inhibitory action lies at a point after cAMP accumulation.
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