Fat Segmentation Research Articles

Growth hormone and dexamethasone stimulate lipolysis and activate adenylyl cyclase in rat adipocytes by selectively shifting Gi alpha2 to lower density membrane fractions.

GH, in the presence of glucocorticoid, produces a delayed increase in lipolysis in rat adipose tissue, but the biochemical mechanisms that account for this action have not been established. Other lipolytic agents rapidly activate adenylyl cyclase (AC) and the resulting production of cAMP initiates a chain of reactions that culminates in the activation of hormone-sensitive lipase. We compared responses of segments of rat epididymal fat or isolated adipocytes to 30 ng/ml GH and 0.1 microg/ml dexamethasone (Dex) with 0.1 ng/ml isoproterenol (ISO), which evoked a similar increase in lipolysis. All measurements were made during the fourth hour after the addition of GH+Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant increases in cAMP were discernible in homogenates of GH+Dex-treated tissues, Rp-cAMPS (Rp-adenosine 3'5'-phosphothioate), a competitive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH+Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32P from [gamma-32P]ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH+Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH+Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K x g or a less dense membrane pellet sedimented at 100K x g, but doubled the AC activity in the 16K pellet when assayed in the absence of forskolin. To evaluate possible effects on G proteins, pellets obtained from centrifugation of adipocyte homogenates at 16K x g and 100K x g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Gi alpha2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gs alpha in the 16K pellet was unaffected by GH+Dex and was decreased (P < 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH+Dex-dependent shift of Gi alpha2 to less dense fractions as determined by both Western analysis and [32P]NAD ribosylation catalyzed by pertussis toxin. No such changes were seen in the distribution of Gs alpha or 5'-nucleotidase. Colchicine (100 microM) blocked the GH+Dex-dependent shift of Gi alpha2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH+Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Gi alpha2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

Transplantation of Purified Autologous Fat

The idea of autologous fat microtransplants has recently resurfaced because of interest by the press. Past experiments have shown that small amounts of fat may be transplanted with an expected survival rate between 10 and 50 percent without ectodermis. A great fund of knowledge exists showing that skin grafts will survive quite dependably if their thickness is up to about 0.0020 inch. For the first week or so, they live by diffusion and inosculation, and then neovascularization enables them to continue viability. I have developed a technique whereby fat is harvested through a blunt cannula with minimal vacuum to prevent explosion of the cellular fat globules. Fibrin and cellular debris are removed, and the fat globules are separated from the free fat, blood, and other constituents of the aspirate. Individual, free-floating 1- to 3-mm adipose fragments are then suspended in nutrient solution and injected through an 18-gauge needle. Multiple radial pathways, with each fat segment being separated from the other by host tissue, maximizes the host-prosthesis interface and the possibility for exchange of nutrients. I have attempted this procedure in more than 100 patients with widely varying results. In acne pits of the face, no significant improvement could be noted 6 weeks after injection. For the first few weeks, a near-perfect result was obtained, but none of these transplants in scarred areas of this kind have been of benefit. Injection of 10 to 50 cc in other areas has resulted in some cells (perhaps 10 percent) surviving over 2 years.(ABSTRACT TRUNCATED AT 250 WORDS)

The Isoquinoline Sulfonamide Inhibitors of Protein Phosphorylation, H-7, H-8, and HA-1004, Also Inhibit RNA Synthesis: Studies on Responses of Adipose Tissue to Growth Hormone*

To evaluate the possibility that some of the metabolic effects of GH in rat adipose tissue depend upon phosphorylation-dephosphorylation reactions, we examined the effects of the isoquinoline sulfonamide family (H-7, H-8, and HA-1004) of protein kinase inhibitors on the actions of GH. In the course of these studies it became clear that these compounds may also block RNA synthesis. In the concentration range of 50-200 microM, H-7, H-8, and HA-1004 completely blocked lipolysis in response to the combination of 100 ng/ml dexamethasone and 30 ng/ml human GH in segments of epididymal fat from normal rats, but were less effective in blocking lipolysis in response to either 1 mM (Bu)2cAMP or 1 ng/ml isoproterenol, which are known to depend upon activation of protein kinase-A. Activation of protein kinase-C with phorbol myristate nearly doubled the rate of glucose oxidation in segments of normal adipose tissue, and this insulin-like response was completely inhibited with 200 microM H-7. At concentrations as high as 500 microM, H-7, H-8, and HA-1004 failed to inhibit the insulin-like response to GH in tissue segments of either normal or hypophysectomized rats. However, when 200 microM H-7 or H-8, but not HA-1004, was present during the first 3 h of treatment with GH, it prolonged the duration of the insulin-like response (acceleration of glucose oxidation) from its normal termination within 2-3 h to more than 4 h. Identical results were obtained with 5 micrograms/ml actinomycin-D. The effect of H-7 or H-8 was reversible and required the continuous presence of these agents, whereas actinomycin-D was required only during the first 60 min after GH. Termination of the insulin-like response normally is followed by a period of several hours in which the tissues are refractory to further insulin-like stimulation by GH. When actinomycin-D, H-7, H-8, or HA-1004 was added to tissues of hypophysectomized rats 60 min after GH, the insulin-like response terminated at its normal time, but the tissues were not refractory to insulin-like stimulation upon reexposure to GH. These agents also prevented GH from sustaining refractoriness in normal adipose tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute effects of S-carboxymethylated human growth hormone on insulin resistance in the obese ( [formula omitted]) mouse

Chronic treatment of ob ob mice with growth hormone (GH) increases plasma insulin and blood glucose concentrations, and enhances insulin resistance in peripheral tissues. The purpose of the present study was: (1) to determine the length of time required for the development of increased circulating insulin concentration and adipose tissue insulin resistance in response to GH in the ob ob mouse, (2) to examine the relationship between the rise in insulin concentration and the development of insulin resistance, and (3) to test whether the hormone derivative could enhance insulin resistance in isolated adipose tissue when added in vitro. Female ob ob mice were injected intraperitoneally (ip) with either saline or 200 μg of S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative, which lacks significant insulinlike or growth-promoting activities. A threefold increase in plasma insulin concentration was observed three and six hours after RCM-hGH injection, but increased hyperglycemia was evident only after six hours. The in vitro stimulatory effect of insulin on [ 14C]glucose oxidation by parametrial adipose tissue was unchanged at three hours but suppressed six hours following administration of RCM-hGH. When the plasma insulin level of ob ob mice was increased threefold by the administration of neutral protamine Hagedorn (NPH) insulin, the in vitro stimulatory effect of insulin on [ 14C]glucose oxidation by adipose tissue isolated from these animals was not altered, suggesting that insulin-induced receptor or postreceptor changes do not account for the increased insulin resistance produced by RCM-hGH. When segments of parametrial fat were incubated for up to six hours in Krebs-Ringer bicarbonate buffer or cultured for up to 24 hours in Earle's medium 199 in the presence of RCM-hGH or biosynthetic methionyl-hGH (met-hGH), subsequent insulin-stimulated [ 14C]glucose oxidation was not affected, suggesting that GH does not enhance insulin resistance in adipose tissue directly.

Metabolic basis for the diabetogenic action of growth hormone in the obese (ob/ob) mouse.

The ob/ob mouse responds predictably to chronic treatment with large doses of pituitary GH with marked hyperglycemia and decreased glucose tolerance. The purpose of the present study was to characterize the metabolic alterations produced by GH that lead to this diabetogenic response in the ob/ob mouse in order to determine whether this animal might serve as a useful model for the study of the cellular mechanisms involved in the diabetogenic action of GH. Female ob/ob mice were treated sc for 3 days with either saline or 200 micrograms/day S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative lacking significant growth-promoting or insulin-like activities. Six hours before the start of the experiment, the animals were given a sc injection of 2 micrograms dexamethasone and deprived of food. RCM-hGH treatment produced marked increases in fasting blood glucose and plasma insulin concentrations, but had no effect on plasma glucagon or serum insulin-like growth factor I levels. It had no effect on liver glycogen level or in vitro hepatic glucose production in the absence or presence of pyruvate and lactate added to the incubation medium. By contrast, the in vitro stimulatory effects of insulin on [14C] glucose oxidation by isolated soleus muscle or segments of parametrial fat were greatly attenuated by RCM-hGH treatment, without changes in rates of basal glucose oxidation. This change in peripheral tissue responsiveness to insulin does not appear to involve glucose transport, since the in vitro stimulation by insulin of 3-O-[14C]methylglucose transport into isolated diaphragm muscle was not altered by RCM-hGH treatment. Moreover, the RCM-hGH-induced reduction in adipose tissue responsiveness to insulin does not appear to be mediated by a reduction in insulin binding, since [125I]iodoinsulin binding to adipocytes isolated from RCM-hGH-treated mice was similar to that to cells from saline-treated animals. Interestingly, the reduction in responsiveness to insulin seen with segments of adipose tissue from RCM-hGH-treated animals was not found with isolated adipocytes prepared from such tissue by collagenase digestion. These results suggest that the hyperglycemia and glucose intolerance produced in ob/ob mice by chronic GH treatment result primarily from increased peripheral tissue insulin resistance. Therefore, the ob/ob mouse provides a useful model to elucidate the cellular mechanism(s) of this aspect of the diabetogenic action of GH.

Effects of hypophysectomy and cell isolation on the transport of L-arabinose by adipocytes.

Hypophysectomy decreased the basal rate of glucose metabolism in segments of epididymal fat studied in vitro and lowered their maximum capacity to use glucose. However, hypophysectomy changed neither the sensitivity to insulin nor the magnitude of the response when the results were expressed relative to the basal rate of glucose metabolism. Adipocytes isolated from both hypophysectomized and normal rats exhibited a higher basal rate of glucose metabolism than cells remaining in situ in the contralateral tissues, but this consequence of cell isolation was more pronounced for adipocytes of hypophysectomized than normal rats. Glucose metabolism could not be further increased by exposure of the adipocytes of hypophysectomized rats to insulin, whereas insulin produced a 3-to 5-fold stimulation of glucose oxidation in normal adipocytes. The effects of insulin and hypophysectomy on the transport of the nonmetabolizable sugar L-[1-14C]arabinose in tissue segments and isolated adipocytes were also studied. Uptake of L-arabinose was usually more rapid in segments of epididymal fat of normal rats than in segments of tissue obtained from hypophysectomized rats and was significantly accelerated by insulin in both groups. Uptake of L-arabinose was more rapid than normal in adipocytes isolated from hypophysectomized rats and, like glucose metabolism, could not be accelerated by insulin. The same concentration of insulin markedly promoted arabinose uptake in normal adipocytes. Efflux of L-arabinose from segments of tissue from hypophysectomized rats was twice as rapid as that from normal tissue and, in contrast with the rate of efflux from normal tissues, was not accelerated by insulin. The data suggest that in the absence of pituitary secretion, sugar transport in the adipocyte membrane may be asymmetrical. The data also support the view that hypophysectomy renders adipocytes more susceptible than normal to the cell isolation procedure which maximally accelerates glucose utilization and inward transport of arabinose in these cells.

Biological activity of bacterial derived human growth hormone in adipose tissue of hypophysectomized rats.

Segments of epididymal fat from hypophysectomized rats were incubated in Krebs-Ringer bicarbonate buffer for 1-4 h. Responses to three bacterially synthesized human GH (hGH) preparations were compared with responses to two highly purified pituitary hGH preparations. Hormones of both bacterial and pituitary origin increased the rate of oxidation of [U14C] glucose to 14CO2 and antagonized epinephrine-stimulated lipolysis. These insulin-like effects were produced over a range of 30-300 ng/ml hGH, and their concentration dependence was quite similar regardless of the source of hormone. Exposure of tissues to GH in the first hour of incubation makes them refractory to the insulin-like actions of a test dose of GH added in the fourth hour of incubation. hGH of either pituitary or bacterial origin also appeared to be equipotent in producing such refractoriness. The minimal effective concentration for this response fell between 10 and 30 ng/ml. Hormones from both sources were also capable of displacing 125I-labeled hGH from specific binding sites on isolated adipocytes, and the displacement curves were superimposable. Fifty percent displacement was seen at concentrations in the range of 10-20 ng/ml. Cell preparations produced a delayed increase in lipolysis which was evident in the presence of 0.3 mg/ml of theophylline in the fourth hour of incubation. In this regard, two of the bacterial hGH preparations were more potent than pituitary hGH and produced statistically significant increases in lipolysis at concentrations 10-30 times lower than pituitary hGH studied simultaneously in tissues of the same rats. Despite the presence of less than 1% contamination with bacterial protein, the greater potency of the bacterial hGH may be due to material of bacterial origin, since one bacterial hGH preparation, which was further purified to remove 99% of the remaining bacterial contamination was equipotent with pituitary hGH. These data indicate that a protein hormone synthesized in bacteria, and therefore likely to be free of peptides which might contaminate hGH purified from pituitary extracts, has all of the diverse physiological effects in adipose tissue that have been observed with preparations of GH derived from pituitary glands. These findings lend strong support to the conclusion that both insulin-like and lipolytic actions of GH are intrinsic properties of the molecule and cannot be attributed to contaminants present in the pituitary extract.

Lipolytic effects of insulin in adipocytes isolated from hypophysectomized rats.

Adipocytes isolated from the epididymal fat of hypophysectomized rats by digestion with collagenase failed to respond to insulin with an increase in glucose utilization. These cells also exhibited an anomalous response to insulin when added in the presence of epinephrine. While insulin antagonized the lipolytic actions of epinephrine in normal adipocytes or in segments of epididymal fat from normal or hypophysectomized rats, it potentiated lipolysis in adipocytes isolated from hypophysectomized rats. This anomalous effect was also evident when isoproterenol, ACTH, or glucagon was used as the lipolytic agent, but the expected antilipolytic response was obtained when theophylline served as the lipolytic agonist. The lipolytic effects of insulin were not seen until 4-7 days after hypophysectomy. Treatment of hypophysectomized rats with a combination of GH (100 micrograms/rat . day), cortisone acetate (1 mg/rat . day), and T3 (1 micrograms/rat . day) for 5 days restored the antilipolytic response to insulin in cells of hypophysectomized rats, but no one hormone alone was effective. The data indicate that adipocytes of hypophysectomized rats retain their ability to recognize and response to insulin, but the ability of insulin to stimulate glucose oxidation or antagonize epinephrine-induced lipolysis is abolished by the cell isolation procedure. The findings underscore the need to consider the impact of hormonal status on the ability of cells to retain normal responsiveness during the rigors of the cell isolation procedure and suggest that failure to do so might lead to erroneous interpretations of the physiological actions of hormones.

Effects of growth hormone on adipose tissue of weanling rats.

Epididymal and parametrial adipose tissues were exised from weanling rats, aged 18–35 days, and tested for responsiveness to GH in vitro. Segments of epididymal fat from rats of the Charles River strain, ranging from 20–32 days of age, responded to GH with an increase in glucose utilization. Tissues from younger rats (18 days old) or older rats (33–35 days old) were unresponsive. Similar results were obtained with rats of the Fisher strain, except that responsiveness of epididymal fat to GH terminated before day 29, while responsiveness of parametrial fat persisted through day 30. Stimulation of glucose metabolism was not always accompanied by increased leucine oxidation, and no antilipolytic effect of GH was ever seen. The insulinlike response, although limited to glucose utilization, was otherwise similar to insulin-like responses seen in adipose tissue of older hypophysectomized rats; it persisted for only 1–2 h and was followed by a period of refractoriness. The minimal concentration of GH needed to p...

Separation of early and late responses of adipose tissue to growth hormone.

GH produces a variety of effects in adipose tissue obtained from hypophysectomized rats. A specific antiserum to rat GH produced in a rhesus monkey was used as a tool to study the relationship of these responses to each other. In all, five different metabolic responses produced by adding 1 microgram/ml rat GH to segments of epididymal fat obtained from hypophysectomized rats were studied. Three of these, stimulation of [U-14C]glucose oxidation to 14CO2, stimulation of L-[1-14C]leucine oxidation to 14CO2, and inhibition of epinephrine-induced lipolysis, are insulin-like. They begin after lag periods of about 25, 35, and 15 min, respectively, and disappear by about 180 min. The addition of antiserum simultaneously with the hormone or within the first 10 min thereafter blocked all three responses. When added more than 10-15 min after GH the antiserum failed to block insulin-like responses measured 30-60 min later, even if the response was prolonged into the fourth hour by adding actinomycin D along with GH to block the cellular restorative processes. Thus, insulin-like effects, once triggered, appear to be independent of the continued presence of the hormone. Furthermore, differences in the lag periods required for each insulin-like response appear to be due to the times required to carry out postreceptor events. Delayed lipolysis, observable in the presence of theophylline, becomes evident only after at least 1 h and can also be blocked by the simultaneous addition of hormone and antiserum. The tissue-hormone interaction which initiates this effect occurs rapidly, and its appearance in the second hour of incubation cannot be blocked if the antiserum is added 5 min after GH. This response persists for at least 5 h. The addition of antiserum 60 or 120 min after GH aborts the lipolytic response, but only after a delay of about 1 h. Thus, unlike the insulin-like responses, the accelerated rate of lipolysis requires the continued presence of the hormone to persist. After the insulin-like effects of GH have run their course, a second insulin-like response cannot be initiated for many hours by a second exposure to GH. The initial exposure to GH renders tissues refractory to the insulin-like action of the hormone. The induction of refractoriness, as measured in the fourth hour, can be blocked by the addition of antiserum simultaneously with GH or as much as 60 min after the hormone, suggesting that prolonged contact between tissue and hormone is required. These studies suggest that GH interacts with adipose tissue in at least three different ways, and that not all of the various responses of tissue to hormone are causally related.

Altered profiles of biological activity of growth hormone fragments on adipocyte metabolism.

A highly purified preparation of human GH (hGH) and two large fragment complexes (Da1 and Dc2) isolated from plasmin digests of reduced and S-carbamidomethylated hGH were tested for their ability to produce five different biological responses in segments of epididymal fat obtained from hypophysectomized rats. Judging by the minimal concentration of hormone needed to produce a statistically significant response, hGH was 3-10 times as potent as Da1 and Dc2 in increasing the oxidation of [U-14C]glucose to 14CO2, hGH and Da1 were equipotent in stimulating the oxidation of L-[1-14C]leucine to 14CO2, antagonizing the lipolytic effect of epinephrine, and inducing refractoriness to the insulin-like action of hGH. At least 3 times as much Dc2 was required to produce significant responses. Da1 was 10 times more active than hGH in producing delayed lipolysis in the presence of theophylline and 30 times as effective as Dc2. These findings suggest that the various responses to GH that can be measured in adipose tissue may result from more than one kind of interaction between GH and adipocytes. (Endocrinology 108: 553, 1981)

Insensitivity of adipocytes of hypophysectomized rats to growth hormone and insulin.

SummaryFree fat cells prepared from epididymal fat of hypophysectomized rats by digestion with collagenase failed to exhibit increased glucose oxidation when exposed to insulin or growth hormone. Segments of epididymal fat from the same hypophysectomized rats responded normally to these hormones. When obtained from normal rats, both segments of epididymal fat and free adipocytes also exhibited normal sensitivity to insulin. Hormonal responsiveness of adipose cells of hypophysectomized animals was not restored by shortening the digestion period, addition of trypsin or phospholipase inhibitors or pretreatment of the donor animals with cortisone, triiodothyronine, or growth hormone.

Permissive effects of hormones on lypolysis.

Hypophysectomy severely reduced glycerol and free fatty acid (FFA) production by segments of epididymal fat. Preincubation of adipose tissue from either hypophysectomized or normal rats with dexamethasone (.016 μg/ml) increased glycerol and FFA production in response to low concentrations of epinephrine (.01–0.05 μg/ml). This effect of dexamethasone had a lag period in excess of 2 hr, and was blocked by actinomycin d. Glucose was not required. Even greater potentiation was seen when growth hormone was also present. It is suggested that acute effects of dexamethasone on lipolysis may result from some modification in the metabolism of cyclic adenosine monophosphate, secondary to the induction of synthesis of some new species of protein. Pretreatment of hypophysectomized rats with triiodothyronine (T3, 5 μg/rat) also markedly potentiated the lipolytic response of segments of adipose tissue to epinephrine and theophylline when tested in vitro 16 hr later. This effect of T3 was not inhibited by actinomycin d. ...

The effects of epinephrine on glycerol production in segments of adipose tissue preincubated with dexamethasone and growth hormone.

SummaryAfter preincubation with dexamethasone (0.016 μg/ml) and growth hormone (1 μg/ml), segments of epididymal fat were tested for sensitivity to the lipolytic effects of epinephrine. When the data from the entire group of 36 rats were pooled and analyzed, it appeared that dexamethasone and growth hormone significantly increased basal lipolysis, but had no significant effect on epinephrine-induced lipolysis. Tissues from only 13 of the 36 rats had increased basal lipolysis of the order of twofold or greater. When the data from these rats were pooled, it appeared that pretreatment with dexamethasone and growth hormone actually reduced lipolysis in response to 0.001 μg/ml of epinephrine and had no effect on higher concentrations of epinephrine. Pooling the data from the remaining 23 rats indicated that the preincubation procedure did not increase basal lipolysis, but significantly increased lipolysis in response to 0.001 and 0.01 μg/ml of epinephrine. The difference in the response of the two groups of ra...

The effects of insulin on lipolysis evoked by cyclic AMP and its dibutyryl analog.

SummaryGlycerol production by segments of epididymal fat from normal rats was used as a measure of lipolysis. Theophyl-line (0.3 mg/ml) produced a striking increase in lipolysis. Further addition of cyclic adenosine 3′, 5′-monophosphate (CAMP) at a concentration of 10-2M caused an even greater increase in glycerol production. Insulin (1 mU/ml) slightly reduced the lipolytic effects of theophylline and completely abolished lipolysis in response to CAMP. The dibutyryl analog of CAMP (10-3M) also increased lipolysis and its effects were also potentiated by theophylline. Insulin failed to reduce and in some experiments even enhanced the lipolytic action of dibutyryl CAMP. Imidazole reduced lipolysis in response to both nucleotides. The findings indicate that CAMP and its dibutyryl analog may behave quite differently under some circumstances and underscore the need for caution in drawing physiological conclusions based on findings with DBCAMP alone.

Metabolic effects of imidazole in adipose tissue

Addition of imidazole (10 mg/ml) to segments of rat epididymal fat reduced the lipolytic response to epinephrine and theophylline and abolished lipolysis in response to cyclic 3',5'-adenosine monophosphate. Basal lipolysis was undiminished. Imidazole also prevented the increase in glucose oxidation caused by epinephrine, but had little effect on glucose oxidation caused by insulin. Imidazole inhibited the incorporation of glucose carbons into long-chain fatty acids and glyceride-glycerol. The data are thought to support the suggestion that imidazole stimulates the activity of phosphodiesterase.

Multiple Effects of Growth Hormone on Lipolysis

Segments of epididymal fat from normal and hypophysectomized rats were incubated in vitro for 4 hr in the presence of growth hormone (0.01–1.0 μg/ml) and the synthetic glucocorticoid, dexamethasone (0.16 μg/ml). The 2 hormones in conjunction increased the release of glycerol and free fatty acids (FFA) from the fat of normal rats but had no effect on lipolysis when tissues of hypophysectomized rats were studied. The addition of theophylline (0.9 mg/ml) to the incubation medium evoked maximal lipolysis in the normal tissues whether or not the hormones were present. The same concentration of theophylline increased the basal lipolysis in the adipose tissues of hypophysectomized rats and caused these tissues to respond to dexamethasone and growth hormone in a manner that was quantitatively and qualitatively similar to the response of normal tissues in the absencs of theophylline. Pretreatment of hypophysectomized rats with L-triiodothyronine (5 μg/rat/day for 2 days) or cortone acetate (2.5 mg/rat/day for 2 da...