Effects of selenium on apoptosis and abnormal amino acid metabolism induced by excess fatty acid in isolated rat hepatocytes.

SCOPE: Increased serum free fatty acid (FFA) occurs in subjects with non-alcoholic fatty liver disease (NAFLD) and also triggers oxidative stress, apoptosis, and insulin resistance. Selenium (Se) is an antioxidant agent. However, the effect of Se on NAFLD or diabetes is still unclear. We investigated the effect of Se on apoptosis and abnormal amino acid metabolism initiated by excess FFA in isolated rat hepatocytes. METHODS AND RESULTS: Primary hepatocytes from rats were isolated and exposed to excessive FFA (0.5 mM oleate/palmitic acid 2:1) and 0.1 µM Se. Se protected primary hepatocytes against oxidative stress and apoptosis induced by excess FFA, but did not play a role on abnormal amino acid metabolism and insulin resistance initiated by FFA in isolated rat hepatocytes. CONCLUSION: Although Se had the capability of preventing the apoptosis initiated by ROS, insulin resistance failed to be reversed in hepatocytes exposed to FFA. This failure may be attributed to the limitation of Se in regulating branched chain amino acids abundance. This indicates that apoptosis and insulin resistance might be involved in different pathways when isolated hepatocytes were exposed to FFA and Se.

Branched-Chain Amino Acid-Rich Supplements Containing Microelements Have Antioxidant Effects on Nonalcoholic Steatohepatitis in Mice.

BACKGROUND: The aim of the present study was to elucidate whether the administration of antioxidant-rich nutrients, including branched-chain amino acids (BCAAs), microelements, and vitamins, both alone and in combination, has a positive impact on liver function in a nonalcoholic steatohepatitis (NASH) mouse model and identify the mechanisms underlying these effects. METHODS: Seven-week-old male KKAy mice fed a methionine- and choline-deficient diet (MCD) for 4 weeks were divided into 7 groups and fed the following planned diets for another 4 weeks: group A (normal diet), group B (MCD; control), group C (MCD with rich microelements), group D (MCD with rich BCAAs), group E (MCD with rich microelements and BCAAs), and group F (MCD with rich microelements, BCAAs, and vitamins). We then conducted biochemical assays, histological analyses, immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2'-nonenal (4-HNE), and Western blotting for insulin glucose signaling, lipid metabolism, and endoplasmic reticulum (ER) stress-related signaling in liver specimens obtained from mice in each group. RESULTS: The morphometric grades of all NASH-related findings and the mean degree of 8-OHdG immunolocalization in groups D-F were significantly lower than those observed in group B. The expression levels of insulin receptor ß subunit (IRß) and p-elF in groups E and F and those of phosphatidyl-inositol 3 kinase (PI3K85), p-AcelCoA, and PERK in group F were similar to those noted in group A. CONCLUSIONS: The administration of a combination of antioxidant-rich nutrients, including BCAAs and microelements, is likely to suppress the progression of NASH by reducing oxidative stress, primarily via the downregulation of the ER stress pathway.

The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 µg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Carbohydrate-to-fat ratio affects food intake and body weight in Wistar rats.

The aim of the study was to evaluate the impact of carbohydrate-to-fat ratio on body weight and appetite regulation in Wistar rats. Twenty-four Wistar rats were randomized to three dietary groups (n = 8): normal carbohydrate diet (NC), low-carbohydrate diet (LC) and high-carbohydrate diet (HC) for 12 weeks. Body weight and food intake were recorded. Circulating leptin and insulin levels were measured by radioimmunoassay method. The expression levels of leptin receptor, insulin receptor, orexin, neuropeptide Y (NPY), agouti-related protein (AgRP) and melanocortin-4 receptor (MC-4R) in the hypothalamus were also measured by realtime polymerase chain reaction (PCR). In the LC group, food intake reduced while body weight increased significantly compared with the NC and HC groups. Plasma leptin levels increased in the LC (18.5 +/- 8.2 ng/mL) group compared with the NC (8.6 +/- 3.8 ng/mL, P < 0.001) and HC (6.6 +/- 1.9 ng/mL, P < 0.001) groups. Realtime reverse transcription-PCR revealed a decrease in the hypothalamic expression level of only leptin receptor in the LC (0.764, 0.471-4.648 copy/mL) and HC (0.357, 0.129-0.781 copy/mL) groups compared with the NC (1.323, 0.616-2.392 copy/mL; P = 0.01) group, and that there was no significant change in those of insulin receptor, AgRP, Orexin, NPY and MC-4R. Low-carbohydrate, high-fat diet raised body weight, which led to a rising of circulating leptin levels and a reduced expression of leptin receptor in the hypothalamus.

Berberine lowers blood glucose in type 2 diabetes mellitus patients through increasing insulin receptor expression.

Our previous work demonstrated that berberine (BBR) increases insulin receptor (InsR) expression and improves glucose utility both in vitro and in animal models. Here, we study the InsR-up-regulating and glucose-lowering activities of BBR in humans. Our results showed that BBR increased InsR messenger RNA and protein expression in a variety of human cell lines, including CEM, HCT-116, SW1990, HT1080, 293T, and hepatitis B virus-transfected human liver cells. Accordingly, insulin-stimulated phosphorylations of InsR beta-subunit and Akt were increased after BBR treatment in cultured cells. In the clinical study, BBR significantly lowered fasting blood glucose (FBG), hemoglobin A(1c), triglyceride, and insulin levels in patients with type 2 diabetes mellitus (T2DM). The FBG- and hemoglobin A(1c)-lowering efficacies of BBR were similar to those of metformin and rosiglitazone. In the BBR-treated patients, the percentages of peripheral blood lymphocytes that express InsR were significantly elevated after therapy. Berberine also lowered FBG effectively in chronic hepatitis B and hepatitis C patients with T2DM or impaired fasting glucose. Liver function was improved greatly in these patients by showing reduction of liver enzymes. Our results confirmed the activity of BBR on InsR in humans and its relationship with the glucose-lowering effect. Together with our previous report, we strongly suggest BBR as an ideal medicine for T2DM with a mechanism different from metformin and rosiglitazone.

Effect of type-1 diabetes mellitus on the regulation of insulin and endothelin-1 receptors in rat hearts.

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.

Adiposity associated rise in leptin impairs ovarian activity during winter dormancy in Vespertilionid bat, Scotophilus heathi.

The aim of the study was to evaluate the seasonal variation in serum leptin levels in a natural population of the female bat, Scotophilus heathi and their relationship to the changes in the body mass, serum insulin level, and ovarian activity. Circulating leptin level varied significantly over the season and correlated positively with the changes in body mass, and circulating insulin and androstenedione (A4) levels. Circulating leptin concentrations showed two peaks; one coincides with the maximum fat accumulation prior to winter dormancy, whereas the second shorter peak coincides with late pregnancy. The in vivo study in S. heathi showed that the increased circulating leptin level during winter dormancy coincides with the decreased expression of ovarian steroidogenic acute regulatory (StAR) protein, and low circulating estradiol (E2) level. At the same time, increased circulating leptin level coincides with increased expression of ovarian insulin receptor and high circulating A4 level. The low circulating leptin level during preovulatory period coincides with the increase in StAR protein but decrease in insulin receptor protein. The in vitro study confirmed the in vivo observations of inhibitory effect of leptin on LH induced StAR expression and E2 production, whereas the stimulatory effect of leptin (high dose) on LH induced expression of insulin receptor protein and A4 production. However, pharmacological dose of leptin produced inhibitory effect on the expression of insulin receptor protein. The results of the present study thus suggest that high circulating leptin level during winter dormancy promotes adiposity and impairs ovarian activity by suppressing StAR-mediated E2 production as well as by enhancing insulin receptor-mediated A4 synthesis thereby contributing anovulatory condition of delayed ovulation in S. heathi.

Intake of trans fatty acid-rich hydrogenated fat during pregnancy and lactation inhibits the hypophagic effect of central insulin in the adult offspring.

OBJECTIVE: Using rats we examined whether maternal intake of hydrogenated fat rich in trans fatty acids affects brain fatty acid profile, hypothalamic content of insulin receptor and insulin receptor substrate-1 proteins, and the hypophagic effect of centrally administered insulin in 3-mo-old male progeny. METHODS: Throughout pregnancy and lactation, Wistar rats ate isocaloric/normolipidic diets with soybean oil (control) or soybean oil-derived hydrogenated fat (trans diet) as a fat source. Upon weaning, the trans offspring continued on the trans diet (trans group) or were switched to a control diet (trans-control group). RESULTS: Compared with control rats, trans rats had lower brain levels of eicosapentaenoic acid. Compared with trans rats, trans-control rats had increased levels of total polyunsaturated fatty acids and arachidonic acid and decreased levels of trans fatty acids, saturated fatty acids, and monounsaturated fatty acids. Insulin receptor and insulin receptor substrate-1 levels were significantly lower (44% and 38%, respectively) in trans than in control rats. In trans-control rats, insulin receptor was 26% lower (P < 0.05), whereas insulin receptor substrate-1 was 50% lower, than in control rats. Insulin decreased 24-h feeding in control (27%) and trans (38%) rats but failed to do so in trans-control rats. The latter group had increased serum glucose levels. CONCLUSIONS: The data suggest that the early (intrauterine/perinatal) exposure to hydrogenated fat rich in trans fatty acids programmed the hypothalamic feeding control mechanisms. As young adults, only trans-control animals showed loss of insulin-induced hypophagia, indicating that the mismatch between early and later nutritional environments was relevant. However, the trans group also showed signs of altered appetite signaling mechanisms, suggesting that the early adaptations may have deleterious consequences later in life.

Pinitol supplementation does not affect insulin-mediated glucose metabolism and muscle insulin receptor content and phosphorylation in older humans.

This study assessed the effect of oral pinitol supplementation on oral and intravenous glucose tolerances and on skeletal muscle insulin receptor content and phosphorylation in older people. Fifteen people (6 men, 9 women; age 66 +/- 8 y; BMI 27.9 +/- 3.3 kg/m(2); hemoglobin A1c 5.39 +/- 0.46%, mean +/- SD) completed a 7-wk protocol. Subjects were randomly assigned to groups that during wk 2-7 consumed twice daily either a non-nutritive beverage (Placebo group, n = 8) or the same beverage with 1000 mg pinitol dissolved into it (Pinitol group, n = 7, total dose = 2000 mg pinitol/d). Testing was done at wk 1 and wk 7. In the Pinitol group with supplementation, 24-h urinary pinitol excretion increased 17-fold. The fasting concentrations of glucose, insulin, and C-peptide, and the 180-min area under the curve for these compounds, in response to oral (75 g) and intravenous (300 mg/kg) glucose tolerance challenges, were unchanged from wk 1 to wk 7 and were not influenced by pinitol. Also, pinitol did not affect indices of hepatic and whole-body insulin sensitivity from the oral glucose tolerance test and indices of insulin sensitivity, acute insulin response to glucose, and glucose effectiveness from the intravenous glucose tolerance test, estimated using minimal modeling. Pinitol did not differentially affect total insulin receptor content and insulin receptor phosphotyrosine 1158 and insulin receptor phosphotyrosine 1162/1163 activation in vastus lateralis samples taken during an oral-glucose-induced hyperglycemic and hyperinsulinemic state. These data suggest that pinitol supplementation does not influence whole-body insulin-mediated glucose metabolism and muscle insulin receptor content and phosphorylation in nondiabetic, older people.

Expression of insulin-like growth factors (IGF)-1 and -2, IGF-binding proteins-2 and -3, and receptors for growth hormone, IGF type-1 and -2 and insulin in the gastrointestinal tract of neonatal calves.

Growth hormone (GH), insulin-like growth factors (IGFs) and insulin influence post-natal gastrointestinal development and function. We have measured by real-time PCR the mRNA levels of IGF-1 and -2, IGF-binding proteins (IGFBPs)-2 and -3, and receptors for GH, IGF type-1 and -2, and insulin in esophagus, rumen, fundus, pylorus, duodenum, jejunum, ileum and colon of calves on days 1 and 5 of life. Levels of mRNA of measured traits were different (P < 0.05) at different gastrointestinal sites. Furthermore, mRNA levels of IGFs, IGFBPs and of receptors for GH and IGF type-1 and -2 and insulin differed (P < 0.05) on days 1 and 5. Differences in mRNA abundance of IGFs, IGFBPs and of receptors for GH, IGFs, and insulin among gastrointestinal sites on days 1 and 5 of life suggest site-specific functional importance and demonstrate that changes are the consequence of ontogenetic development and/or due to feeding.

Feeding different amounts of colostrum or only milk replacer modify receptors of intestinal insulin-like growth factors and insulin in neonatal calves.

Colostrum intake influences growth and development of the gastrointestinal tract (GIT) in several species and colostral insulin-like growth factors I and II (IGF-I and IGF-II), and insulin are involved in neonatal intestinal tissue growth. We have studied IGF type 1, IGF type 2, and insulin receptors in the intestine of 8-day-old calves fed different amounts of colostrum or only milk replacer. Calves were fed colostrum of the first six milkings on the first 3 days and then milk replacer (GrC(6)) or colostrum only once and then milk replacer (GrC(1)) or they were fed only milk replacer from the beginning, i.e., no colostrum (GrM). Competitive binding studies and ligand blots confirmed the presence of IGF type 1, IGF type 2, and insulin receptors in mucosal cell membranes of duodenum, jejunum, ileum, and colon. The IGF type 1 receptor number in ileum and total intestine in GrC(6) was greater (P<0.05) than in GrC(1) and in GrM, and IGF type 2 receptor number in total intestine was greater (P<0.05) in GrC(6) than in GrM. Insulin binding was best fitted by a model with two binding sites. High affinity insulin receptor numbers in duodenum, ileum, and total intestine were greater (P<0.05) in GrC(6) than in GrM. The data demonstrate that IGF type 1, IGF type 2, and insulin receptors in intestinal mucosa of neonatal calves are influenced by feeding.

mRNA of insulin-like growth factor (IGF) quantification and presence of IGF binding proteins, and receptors for growth hormone, IGF-I and insulin, determined by reverse transcribed polymerase chain reaction, in the liver of growing and mature male cattle.

Plasma insulin-like growth factor-I (IGF-I) concentrations were related to hepatic levels of IGF-I mRNA measured by competitive reverse transcription polymerase chain reaction (PCR) (RT-PCR) in neonatal (8 d old) calves, veal calves, fattened castrated bulls and mature intact bulls. Furthermore, the presence of mRNAs of IGF-II and of receptors for IGF-I (IGF-IR), growth hormone (GHR) and insulin (IR), as well as mRNAs of IGF binding proteins (IGFBP-1, -2 and -3) were assessed by RT-PCR. Hepatic IGF-I mRNA levels and plasma IGF-I concentrations in veal calves, fattened castrated bulls and in intact bulls were 4 to 8 times higher than in 8-d old calves and were 2 to 3 times higher in calves fed colostrum than in calves fed only milk replacer. Hepatic IGF-I mRNA concentrations were closely correlated (r = 0.92) with plasma IGF-I concentrations, suggesting that hepatic IGF-I production largely determines plasma IGF-I levels. The presence of IGF II, IGF-IR, GHR, IR and IGFBP-1, -2 and -3 mRNA was confirmed in the liver of 8-d old calves and older cattle as well, and among newborn calves their presence was independent of differences in nutrition. In conclusion, the major hepatic components of the GH-IGF axis were present in neonatal calves, but the IGF-I expression and therefore also plasma IGF-I levels were relatively low.

Dietary fish oil and olive oil improve the liver insulin receptor tyrosine kinase activity in high sucrose fed rats.

In order to shed light on the possible beneficial effect of dietary unsaturated fatty acids on insulin binding, the effect of fish oil and olive oil administration on insulin binding, autophosphorylation and tyrosine kinase activity of partially purified liver insulin receptors were investigated. These data were confronted with the parameters of sugar and lipid metabolism (blood glucose, insulin and triglycerides), with liver plasma membrane fluidity and fatty acid composition. High sucrose feeding resulted in the elevation of blood glucose and triglyceride level, while the supplementation of animals with fish oil reduced that of triglycerides and olive oil that of insulin. Any significant changes between experimental groups were not detected either in insulin binding to partially purified liver insulin receptor nor in receptor autophosphorylation. However, the insulin stimulated tyrosine kinase activity towards an exogenous substrate (poly(Glu,Tyr)) was decreased by about 50% in the receptors solubilized from liver membranes of sucrose fed rats. Increased dietary intake of fish oil or olive oil restored the activity of insulin tyrosine kinase towards control values, half maximal effect being obtained at similar insulin concentration in all groups. Such improvement might be due to the induced increase of membrane fluidity by unsaturated fatty acids, and/or to the decrease of insulinemia.

Immunohistochemical localization of insulin receptors and phosphotyrosine in the brainstem of the adult rat.

Previous studies have demonstrated that insulin receptors are widely distributed throughout areas of the forebrain in the adult rat that are involved in modulating neuroendocrine functions and feeding behaviour. In addition, a recent investigation showed that there is a good correlation between the presence of the insulin receptor and phosphotyrosine-containing proteins in these regions, indicating a possible functional activity of insulin receptors in vivo. It is unknown whether neural connections between specific brainstem nuclei to forebrain regions may also be under direct regulation of insulin or related factors. In order to test this possibility, the distribution of insulin receptors and phosphotyrosine was mapped throughout the hindbrain of the adult rat by immunocytochemistry, using specific antibodies against the beta-subunit of the insulin receptor as well as against phosphotyrosine. Both markers showed a high degree of overlap throughout numerous distinct anatomical regions of the hindbrain. In the mesencephalon, insulin receptor and phosphotyrosine-positive neurons were found in the precommissural nucleus, the lateral and dorsal part of the central gray, the mammillary bodies and the interpeduncular nucleus. In addition, immunoreactivity was found in the subependymal layer around the aqueduct along fibres and nerve cells possibly contacting the cerebrospinal fluid. In the pons and medulla, dense immunoreactivity was seen in the lateral superior olive, nucleus of the solitary tract, spinal trigeminal nucleus and nucleus ambiguous. Scattered cells were found in the pontine and vestibular nuclei, as well as in the reticular formation. The cerebellum contained moderately dense immunoreactivity in the granule cell and molecular cell layer of the cortex, as well as in the deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

The treatment of poorly controlled non-insulin-dependent diabetic subjects with granulated guar gum.

The treatment of poorly controlled, non-compliant non-insulin-dependent diabetic subjects for one month with guar granules (Guarem) was associated with significant improvements in fasting serum glucose and insulin and urinary glucose excretion. No significant change was observed in either oral glucose tolerance, erythrocyte insulin receptor binding, serum calcium, cholesterol, triglyceride or HbA1. Subjects reported significant side effects including excessive flatus, increased bowel frequency and fullness. The limited advantages of Guarem treatment must be measured against the possibility of these side effects which to a large extent may be avoided by special attention to the means of administration. Prudent supplementation of the diet with Guarem has undoubted potential for diabetic control.

Dietary supplementation of omega-3 polyunsaturated fatty acids improves insulin sensitivity in non-insulin-dependent diabetes.

The effect of dietary omega-3 polyunsaturated fatty acids on the lipid composition and fluidity of erythrocyte membranes, and on in vivo insulin sensitivity was studied in 6 non-insulin-dependent diabetic (NIDD) patients. An 8 weeks daily supplementation of 3 g of the omega 3 fatty acids eicosapentaenoic and docosahexaenoic acid resulted in an increase of the membrane phospholipid unsaturation and the sphingomyelin content. Erythrocyte membrane fluidity, measured with electron spin resonance of intact erythrocytes and with fluorescence polarization of erythrocyte ghosts, did not change. The in vivo insulin stimulated glucose uptake was estimated by determining the metabolic clearance rate (MCR) of glucose in the steady state of a simultaneous infusion during 150 min of glucose (33 mumol/kg/min) and insulin (50 mU/kg/hr). The MCR of glucose increased in all patients; from 3.93 +/- 0.55 - 4.69 +/- 0.74 ml/kg/min (mean +/- SEM, p less than 0.05). Plasma triglyceride concentrations fell from 1.9 +/- 0.3 - 1.2 +/- 0.2 mmol/l (mean +/- SEM, p less than 0.05). We conclude that in NIDDs dietary supplementation of omega 3 fatty acids improves in vivo insulin sensitivity and lowers plasma triglyceride levels, while erythrocyte membrane fluidity remains unaltered.

Insulin receptors on cultured hypothalamic cells: functional and structural differences from receptors on peripheral target cells.

We have studied the functional and structural characteristics of insulin receptors on cultured rat hypothalamic cells. The receptors on these cells are specific for insulin, but have a lower binding affinity than that measured in nonneuronal tissues. Neither acute (2-h) nor long term (24-h) exposure of the hypothalamic cells to high insulin concentrations resulted in receptor down-regulation. However, insulin is internalized in these cells and accumulated in the presence of the lysomotropic agent chloroquine. Acute exposure to insulin does not alter initial rate of 2-deoxyglucose transport in hypothalamic cells, but does cause a stimulation of aminoisobutyric acid uptake. Photoaffinity labeling of the receptors of the hypothalamic cells with a biologically active photosensitive insulin revealed a major specifically labeled band of 115K mol wt and a minor band of 40K mol wt under disulfide-reducing conditions compared to bands of 125K and 90K mol wt seen after labeling of the insulin receptors of adipocytes. The receptor proteins in hypothalamic cells under nonreducing conditions (420K, 370K, and 310K mol wt) were also smaller than those in adipocytes. Thus, the insulin receptors of cultured hypothalamic cells differ from insulin receptors on peripheral target tissues in both functional and structural aspects.

Influence of membrane fluidity changes upon the imprinting of polypeptide hormones in Tetrahymena.

Hormonal imprinting is a physiological phenomenon, in which after the first encounter the receptorial and functional responses of a cell change for future occasions. The present experiments demonstrate (using Tetrahymena as a model cell) that the imprinting is very sensitive to the changes in membrane physical state. Cultivation of Tetrahymena cells in 28 or 15 degrees C or in ergosterol-supplemented media caused only quantitative differences in the imprinting; however, the process of cooling (shift-down) or reheating (shift-up) resulted in a false reaction. The combined treatment by ergosterol and cooling completely abolished the imprinting. These results indicate that hormonal imprinting is a membrane-dependent process.