Leszek Nogowski

Genistein affects lipogenesis and lipolysis in isolated rat adipocytes.

Genistein is a phytoestrogen found in several plants eaten by humans and food-producing animals and exerting a wide spectrum of biological activity. In this experiment, the impact of genistein on lipogenesis and lipolysis was studied in isolated rat adipocytes. Incubation of the cells (10(6) cells/ml in plastic tubes at 37 degrees C with Krebs-Ringer buffer, 90 min) with genistein (0.01, 0.3, 0.6 and 1 mM) clearly restricted (1 nM) [U-14C]glucose conversion to total lipids in the absence and presence of insulin. When [14C]acetate was used as the substrate for lipogenesis, genistein (0.01, 0.1 and 1 mM) exerted a similar effect. Thus, the anti-lipogenetic action of genistein may be an effect not only of alteration in glucose transport and metabolism, but this phytoestrogen can also restrict the fatty acids synthesis and/or their esterification. Incubation of adipocytes with estradiol at the same concentrations also resulted in restriction of lipogenesis, but the effect was less marked. Genistein (0.1 and 1 mM) augmented basal lipolysis in adipocytes. This process was strongly restricted by insulin (1 microM) and H-89 (an inhibitor of protein kinase A; 50 microM) and seems to be primarily due to the inhibitory action of the phytoestrogen on cAMP phosphodiesterase in adipocytes. Genistein at the smallest concentration (0.01 mM) augmented epinephrine-stimulated (1 microM) lipolysis but failed to potentiate lipolysis induced by forskolin (1 microM) or dibutyryl-cAMP (1 mM). These results suggest genistein action on the lipolytic pathways before activation of adenylate cyclase. The restriction of lipolysis stimulated by several lipolytic agents--epinephrine, forskolin and dibutyryl-cAMP were observed when adipocytes were incubated with genistein at highest concentrations (0.1 and 1 mM). These results prove the inhibitory action of this phytoestrogen on the final steps of the lipolytic cascade, i.e. on protein kinase A or hormone sensitive lipase. Estradiol, added to the incubation medium, did not affect lipolysis. It can be concluded that genistein significantly affects lipogenesis and lipolysis in isolated rat adipocytes.

Genistein--a dietary compound inducing hormonal and metabolic changes.

Genistein is a plant-derived compound possessing well-known preventive activity in breast and prostate cancer, cardiovascular diseases and post-menopausal problems. Lately, the interests in genistein have widened. The studies concerning effects of genistein performed on animals and humans revealed other aspects of its action -- the metabolic alterations at the cellular level and in the whole organism. It was shown that genistein decreased body and fat tissue weight gains accompanied by reduced food intake. After ingestion of dietary genistein, the alterations in concentrations of hormones such as: insulin, leptin, thyroid hormones, adrenocorticotropic hormone, cortisol and corticosterone were observed. The changes in lipid parameters -- triglycerides and cholesterol were also noticed as a consequence of genistein administration. Moreover, the altered expression of genes engaged in lipid metabolism, disturbed glucose transport into cells, affected lipolysis and lipogenesis and changed ATP synthesis were found as a result of genistein action.

Genistein-Induced Changes in Lipid Metabolism of Ovariectomized Rats

The effect of the isoflavone, genistein, on the lipid metabolism of ovariectomized rats was studied. Three types of experiments were performed. In the first one, the rats were fed diets supplemented with 0.01 or 0.1% of genistein for 14 days. In the second and third experiments, the direct effect of genistein on the liver and fat tissue were measured respectively by means of liver perfusion or incubation of isolated adipocytes with the isoflavone. Genistein in food significantly decreased blood serum and muscle triglyceride concentrations and increased the level of free fatty acids in serum. Serum free cholesterol was diminished and liver cholesterol was enhanced after genistein ingestion. When genistein acted directly on the liver during perfusion, a smaller incorporation of 14C-glucose into lipids was observed, and in parallel a greater output of free fatty acids into the medium was noticed. These changes were accompanied by diminution of the liver triglyceride contents. Genistein, acting on the adipocytes strongly depressed both basal and insulin-induced lipid synthesis, when glucose was used as a substrate. The effect of the isoflavone alone on the lipolysis in the adipocytes was negligible. However, it intensified lipolysis induced by epinephrine. The results obtained let us conclude that genistein in food can reduce the fattening processes in ovariectomized rats. This effect of genistein may be attributed, at least in part, to its direct influence on lipid metabolism in the liver and adipose tissue.

Genistein restricts leptin secretion from rat adipocytes.

The isoflavones--genistein and daidzein -- compounds found in high concentrations in soy play an important role in prevention of many diseases and affect some metabolic pathways. In the performed experiment it was demonstrated that genistein (5mg/kg b.w.) administered intragastrically for three days to male Wistar rats substantially diminished blood leptin level. Studies with isolated rat adipocytes revealed that this phytoestrogen strongly restricted leptin secretion from these cells. These effects were not accompanied by any changes in leptin gene expression in adipocytes. Daidzein-- an analogue of genistein -- used at similar concentrations did not affect blood leptin concentration, leptin secretion and expression of its gene. To determine the influence of genistein and daidzein on leptin release, adipocytes isolated from the epididymal fat tissue were incubated for 2h in Krebs--Ringer buffer. Leptin secretion stimulated by glucose with insulin was significantly diminished by genistein (0.25--1mM). This effect of genistein may arise from several aspects of its action in adipocytes documented in the literature such as the inhibition of glucose transport and metabolism, the attenuation of insulin signalling, the inhibition of cAMP phosphodiesterase and the stimulation of lipolysis. However, the bypassing of the restrictive action of genistein on glucose transport and glycolysis (by the use of alanine instead of glucose) and on insulin action (by the use of nicotinic acid) was not sufficient to restore leptin secretion from isolated adipocytes. It was also demonstrated that the restriction of the stimulatory influence of genistein on cAMP/protein kinase A (PKA) pathway (by the inhibition of PKA activity) did not improve leptin release. Results obtained in our experiments point at the restriction of glucose metabolism following formation of pyruvate as the pivotal reason of the inhibitory action of genistein on leptin release.

Daidzein, coumestrol and zearalenone affect lipogenesis and lipolysis in rat adipocytes

Daidzein, coumestrol and zearalenone - compounds called phytoestrogens, considered as active biological factors affecting many important physiological and biochemical processes appeared to be also significant regulators of adipocyte metabolism. In our experiments the influence of daidzein (0.01, 0.1 and 1 mM), coumestrol (0.001, 0.01 and 0.1 mM), zearalenone (0.01, 0.1 and 1 mM) and estradiol (0.01, 0.1 and 1 mM) on basal and insulin-stimulated (1 nM) lipogenesis from glucose and acetate was tested in adipocytes isolated from growing (160 +/- 5 g b.w) male Wistar rats. All tested compounds significantly attenuated glucose conversion to lipids. In the case of daidzein and coumestrol, this effect was probably due to inhibition of glycolysis. Daidzein (0.01, 0.1 and 1 mM), coumestrol (0.01 and 0.1 mM) and zearalenone (0.01, 0.1 and 1 mM) affected also basal and epinephrine-stimulated (1 microM) lipolysis. Daidzein (0.01 and 1 mM) augmented basal glycerides breakdown in adipocytes. The epinephrine-induced lipolysis was dependent on daidzein concentration and its stimulatory (0.1 mM) or inhibitory (1 mM) influence was observed. Zearalenone changed lipolysis only at the concentration of 1 mM and its effect was contradictory in the absence or presence of epinephrine (the stimulatory or inhibitory effect, respectively). Results obtained in experiments with inhibitors (insulin, 1 nM and H-89, 50 microM) and activators (dibutyryl-cAMP, 1 mM and forskolin, 1 microM) of lipolysis allowed us to assume that daidzein augmented basal lipolysis acting on PKA activity. The inhibitory effect of daidzein and zearalenone on epinephrine-induced lipolysis is probably due to restriction of HSL action. The influence of coumestrol on glycerides breakdown was less marked. Estradiol augmented only epinephrine-stimulated lipolysis.

Genistein, a plant-derived isoflavone, counteracts the antilipolytic action of insulin in isolated rat adipocytes.

Genistein is a phytoestrogen exerting numerous biological effects. Its direct influence on adipocyte metabolism and leptin secretion was previously demonstrated. This study aimed to determine whether genistein antagonizes the antilipolytic action of insulin in rat adipocytes. Freshly isolated adipose cells were incubated for 90 min with epinephrine, epinephrine with insulin and epinephrine with a specific inhibitor of protein kinase A (H-89) at different concentrations of genistein (0, 6.25, 12.5, 25, 50 and 100 microM). Genistein failed to affect epinephrine-induced glycerol release, however, the inhibitory action of insulin on epinephrine-induced lipolysis was significantly abrogated in cells exposed to the phytoestrogen (12.5-100 microM). The increase in insulin concentration did not suppress the genistein effect. Its inhibitory influence on the antilipolytic action of insulin was accompanied by a substantial rise in cAMP in adipocytes. This rise appeared despite the presence of 10nM insulin in the incubation medium. Further experiments, in which insulin was replaced by H-89, revealed that the antilipolytic action of protein kinase A inhibitor on epinephrine-induced lipolysis was not affected by genistein. This means that genistein counteracted the antilipolytic action of insulin due to the increase in cAMP levels and activation of protein kinase A in adipocytes. The observed attenuation of the inhibitory effect of insulin on triglyceride breakdown evoked by genistein was not related to its estrogenic activities, as evidenced in experiments employing the intracellular estrogen receptor blocker, ICI 182,780. Moreover, it was found that genistein-induced impairment of the antilipolytic action of insulin was not accompanied by changes in the proportion between fatty acids and glycerol released from adipocytes. The ability of genistein to counteract the antilipolytic action of insulin may contribute to the decreased triglyceride accumulation in adipose tissue.

Effect of Isoflavone Genistein on Insulin Receptors in Perfused Liver of Ovariectomized Rats

The experiments were carried out on ovariectomized Wistar rats. Their livers were perfused with basic perfusion medium (BPM) or BPM supplemented with isoflavone genistein, insulin or combination of the two factors. The obtained results support the hypothesis that genistein influences the kinetics of insulin binding to cell membranes changing the number of insulin receptors and dissociation constant (Kd). BPM supplementation with genistein decreased number of high affinity insulin receptors (HAIR) both in livers treated and untreated with insulin. The amount of HAIR diminished significantly from 610 +/- 77 x 10(-15) (no genistein) to 238 +/- 72 x 10(-15) mol/mg of membrane protein (supplement of genistein). Similarly, genistein reduced slightly the amount of HAIR even when added together with insulin (372 +/- 59 x 10(-15) mol/mg) in comparison to rats perfused with medium containing insulin but not the isoflavone (421 +/- 46 x 10(-15) mol/mg). Simultaneously, genistein decreased significantly Kd for HAIR (perfusion with BPM--1.44 +/- 0.18 x 10(-9) mol/l; perfusion with BMP + genistein--0.83 +/- 0.20 x 10(-9) mol/l). Such effects of genistein during liver perfision did not take place when the liver membranes were in vitro incubated with this xenobiotic.

Acute leptin action on insulin blood level and liver insulin receptor in the rat.

Aim of the study was to investigate acute leptin effect on insulin blood level and liver insulin binding in the rat. The administration of leptin induced time and dose dependent decrease in the insulin level, which was statistically significant in comparison to the control animals 120 min after administration of higher dose of peptide (0.30 +/- 0.05 vs 0.14 +/- 0.01 nmol/l, respectively). Simultaneously, we have shown the attenuation of liver sensitivity to insulin 2 hours after higher leptin dose injection. This phenomenon was caused by the decrease of binding capacity of high affinity insulin receptor sites (HAIR), which was statistically significant after higher leptin dose administration at both time points (0.54 +/- 0.13 vs 0.26 +/- 0.03 and 0.71 +/- 0.12 vs 0.40 +/- 0.05 pmol/mg protein for 1 and 2 h, respectively). The present study provides evidence that leptin, in addition to its inhibitory effect on insulin secretion, acts as a modulator of insulin receptor, through the decrease of binding capacity. It seems legitimate to suggest that leptin-induced decrease of insulin receptor binding capacity may be one of several causes of insulin resistance.

Short-time deoxynivalenol treatment induces metabolic disturbances in the rat

Deoxynivalenol (DON) is produced by several Fusarium species and may contaminate feeds causing reduced food consumption and utilisation, reduced body weight gain and other unfavourable changes in animals. To verify if its adverse influence involves also hormonal and metabolic changes, the effect of DON on blood insulin, glucagon, leptin and metabolic parameters in growing Wistar rats was studied. Animals were treated subcutaneously with DON in the amount of 1 mg/kg b.w. After 3 days a significant increase in blood insulin, glucose and free fatty acids were observed in these animals in comparison to the control group. DON treatment caused an increment in glycogen depots and a reduction in triglycerides content in the muscle. Studies on isolated adipocytes revealed that DON (20 micromol/l) slightly stimulated basal lipogenesis, whereas insulin-induced lipid synthesis and lipolysis were unchanged. Results obtained after subcutaneous DON administration indicate that its adverse effects in animals may partially result from metabolic disturbances evoked by this mycotoxin.

Effects of phytoestrogen-coumestrol on lipid and carbohydrate metabolism in young ovariectomized rats may be independent of its estrogenicity.

Two groups of young ovariectomized female rats received one of two treatments. The first group was fed coumestrol in lab chow (200 microg of coumestrol per day) for 14 days; the second group received coumestrol (40 mg/L) via perfusion medium. There was a significant increase (78% compared with the control group) in the uterine weight after coumestrol treatment, which supports the estrogen-like activity of coumestrol. Phytoestrogen diminished the liver and skeletal muscle glycogen contents by 18% and 29%, respectively, and increased the blood glucose level by 24%. Glycogenolytic activity of coumestrol was observed when it acted directly on the liver areas. Although phytoestrogen did not influence insulin and glucagon blood level, liver and to some degree muscle susceptibility to insulin (measured as hormone binding by insulin receptors) was decreased. Coumestrol increased the content of triglycerides in muscle by 113% and enhanced the liver lipid synthesis from glucose by 179%. Liver cholesterol concentration was increased both after coumestrol feeding (by 12%) and when it acted directly on the liver (by 16%). These observations suggest that coumestrol is in general anabolic with regard to lipid and catabolic within-carbohydrate metabolism of young ovariectomized female rats. Based on the results of this study, it is concluded that influence of coumestrol on lipid and carbohydrate metabolism of ovariectomized rats is in part not related to its estrogenic action.