Regulatory effects of baicalin, a flavonoid compound, on adipocyte metabolism.

Baicalin is a plant-derived, biologically active compound exerting numerous advantageous effects. Adipocytes store and release energy in the process of lipogenesis and lipolysis. Rodent studies have shown that baicalin treatment positively affects fat tissue, however, data on the direct influence of this compound on adipocyte metabolism is lacking. In the present research, the short-term effects of 25, 50, and 100 µM baicalin on glucose transport, conversion to lipids, and oxidation, and also on lipolysis in primary rat adipocytes were explored. Lipolysis was measured as glycerol release from adipocytes. It was shown that 100 µM baicalin reduced glucose oxidation but at any concentration did not affect glucose transport and lipogenesis. Baicalin significantly increased the adipocyte response to physiological and pharmacological lipolytic stimuli (such as epinephrine - adrenergic agonist, DPCPX - adenosine A1 receptor antagonist, and amrinone - cAMP phosphodiesterase inhibitor). The stimulatory effects of baicalin on epinephrine-induced lipolysis were markedly diminished by insulin (activator of cAMP phosphodiesterases) and H-89 (PKA inhibitor). It was also demonstrated that baicalin evoked a similar rise in epinephrine-induced lipolysis in the presence of glucose and alanine. Our results provided evidence that baicalin may reduce glucose oxidation and is capable of enhancing lipolysis in primary rat adipocytes. The action on lipolysis is glucose-independent and covers both the adrenergic and adenosine A1 receptor pathways. The rise in cAMP content is proposed to be responsible for the observed potentiation of the lipolytic process.

Distinct dynamics of lipid accumulation by porcine cumulus cells during in vitro maturation with follicular fluid of low and high fatty acid contents.

Follicular fluid (FF) is a complex biological medium providing a fully balanced microenvironment for the oocyte. The standard medium for in vitro maturation of porcine oocytes contains 10% of FF which due to its unknown and inconstant composition is a source of a significant variation. This study aimed to investigate whether follicular fluids of significantly different fatty acid contents (standardized follicular fluid) supplemented to porcine IVM media affects lipid metabolism of porcine oocytes and cumulus cells. Two categories of FF from cyclic gilts containing high (H) and low (L) FA content was added to IVM medium for oocytes from prepubertal gilts. Altogether, 521 cumulus oocyte-complexes (oocytes and corresponding cumulus cells) were analyzed for mRNA expression of 7 genes regulating lipid metabolism and selected traits of the lipid droplets (LD). The applied FFs of different FA levels exerted distinct effects on oocytes and cumulus cells (CCs). During IVM oocytes tended to utilize the lipids as demonstrated by the reduced LD number and lipid fluorescence, whereas cumulus cells accumulated lipids as indicated by the increase in LD number, the occupied area and fluorescence level. Changes in cumulus cells were independent of the FA content in the follicular fluid which means an efficient lipid accumulation during IVM. Final analysis including the effect of FA level on LD traits in oocytes and corresponding CCs revealed two distinct patterns. COCs matured in FF of high FA content were characterized by elevated dynamics of lipid accumulation in CCs and stable lipid content in oocytes. In the case of FF with low FA content, CCs accumulated lipids at a significantly lower rate whereas lipid level in oocytes was reduced. The alterations observed in the LD parameters were not accompanied by changes in oocyte nuclear maturation and in transcript level of any from the 7 analyzed genes. In conclusion, fatty acid content of the follicular fluid supplemented to porcine IVM medium affects lipid metabolism in cumulus cells of the maturing oocyte and application of a standardized FF may help to improve the quality of porcine oocytes matured in vitro.

Betaine supplementation to rats alleviates disturbances induced by high-fat diet: pleiotropic effects in model of type 2 diabetes.

Betaine is a biologically active compound exerting beneficial effects in the organism, however, the exact mechanisms underlying its action are not fully elucidated. The present study aimed to explore, whether betaine alleviates disorders induced by feeding rats a high-fat diet (HFD). Rats were divided into 3 groups: control, fed an HFD and fed an HFD and receiving betaine (2% water solution for 8 weeks). Betaine improved glucose tolerance, decreased blood levels of non-esterified fatty acids and prevented lipid accumulation in the skeletal muscle of rats on an HFD. Betaine reduced activities of blood alanine aminotransferase, blood levels of bilirubin and hepatic lipid content. Expression of fatty acid synthase in the liver and the skeletal muscle was decreased in response to feeding an HFD, and this effect was deepened by betaine in the muscle tissue. Hepatic and muscular expression of genes related to insulin signaling were unchanged in HFD-fed rats. Lipolysis stimulated by epinephrine (an adrenergic receptor agonist), forskolin (an activator of adenylate cyclase), dibutyryl-cAMP (an activator of protein kinase A) and DPCPX (an adenosine A1 receptor antagonist) was diminished in the adipocytes of rats fed an HFD, however, this effect was alleviated by betaine. Moreover, blood leptin levels in HFD-fed rats were elevated, whereas leptinemia have normalized by betaine supplementation. Betaine prevented the increase in expression of N-methyl D-aspartate receptors in the hippocampus and in the cerebral cortex. These results indicate that betaine positively affects the insulin-sensitive tissues: liver (hepatoprotective effects), skeletal muscle (reduced lipid accumulation) and adipose tissue (a rise in lipolysis), which is associated with improved insulin sensitivity. Betaine-induced prevention of hyperleptinemia indicates restoration of leptin action, and changes in the brain reveal neuroprotective properties. Our results show that betaine induces positive changes in HFD-fed rats, its action is pleiotropic and involves different tissues.

Resveratrol reduces excessive cholesterol accumulation in Goto-Kakizaki rat, a model with congenital type 2 diabetes.

Resveratrol (3, 5, 3'-trihydroxystilbene) is a naturally-occurring, biologically active compound having numerous beneficial effects in the organism, including anti-diabetic properties. Its anti-diabetic action have been relatively well established using various animal models, however, in Goto-Kakizaki (GK) rats is poorly explored. These animals are non-obese and have a congenital type 2 diabetes. In the present study, effects of resveratrol on cholesterol content, blood levels of some hormones (thyroxine, triiodothyronine, ghrelin and spexin), glucose and parameters indirectly related with renal function (creatinine, urea nitrogen, total protein and albumin) were explored in GK rats. GK and control rats were treated with resveratrol for 10 weeks at the dose of 20 mg/kg body weight. It was shown that cholesterol content was significantly increased in the blood, liver and the skeletal muscle of diabetic rats, compared with the control animals. However, the resveratrol therapy was associated with a markedly reduced tissue cholesterol content. Our study also demonstrated that blood levels of thyroxine (T4) were decreased, and triiodothyronine (T3) increased in GK rats. These alterations were, however, not significantly affected by resveratrol. GK rats had elevated blood glucose levels, but hyperglycemia was not ameliorated by resveratrol. It was also shown that blood creatinine levels were increased in diabetic rats. However, in animals subjected to the resveratrol therapy, the blood creatinine level was unchanged. Concentrations of ghrelin, spexin and other blood parameters indirectly related with the renal function were shown to be similar in GK and control rats. These results indicate that resveratrol beneficially influences cholesterol concentrations in tissues of diabetic rats; however, it is ineffective in the case of thyroid hormones and glucose. Moreover, it was shown that resveratrol did not induce any significant effects in non-diabetic animals.

Resveratrol alleviates ethanol-induced hormonal and metabolic disturbances in the rat.

Resveratrol is a polyphenol found in different plant species and having numerous health-promoting properties in animals and humans. However, its protective action against deleterious effects of ethanol is poorly elucidated. In the present study, the influence of resveratrol (10 mg/kg/day) on some hormones and metabolic parameters was determined in rats ingesting 10 % ethanol solution for two weeks. Blood levels of insulin, glucagon and adiponectin were affected by ethanol, however, resveratrol partially ameliorated these changes. Moreover, in ethanol drinking rats, liver lipid accumulation was increased, whereas resveratrol was capable of reducing liver lipid content, probably due to decrease in fatty acid synthesis. Resveratrol decreased also blood levels of triglycerides and free fatty acids and reduced gamma-glutamyl transferase activity in animals ingesting ethanol. These results show that resveratrol, already at low dose, alleviates hormonal and metabolic changes induced by ethanol in the rat and may be useful in preventing and treating some consequences of alcohol consumption.

Inhibition of phosphodiesterase 3B in insulin-secreting cells of normal and streptozocin-nicotinamide-induced diabetic rats: implications for insulin secretion.

Cyclic adenosine monophosphate (cAMP) plays important role in the potentiation of insulin secretion in pancreatic B-cells. However, the relevance of cAMP-degrading enzymes in the regulation of insulin secretion is not fully elucidated. The present work was undertaken to determine effects of inhibition of phosphodiesterase 3B (PDE3B) by amrinone on insulin secretion from pancreatic islets and perfused pancreas of normal and mildly diabetic rats. Inhibition of this enzyme was demonstrated to substantially increase insulin-secretory response to 6.7 mM glucose in the isolated islets and perfused pancreas of non-diabetic rats. Increment in glucose-induced insulin secretion resulting from inhibition of PDE3B was accompanied by an increase in islet cAMP levels and was suppressed by inhibition of protein kinase A. It was also demonstrated that insulin secretion stimulated by glucose and 1 µM forskolin was only slightly elevated in the presence of amrinone. Moreover, insulin release induced by succinate instead of glucose was also augmented by inhibition of PDE3B in rat islets. However, exposure of the pancreatic islets of streptozotocin-nicotinamide-induced diabetic rats to amrinone appeared to be without any effect on glucose-induced insulin secretion. Similar lack of response was shown in the perfused pancreas of diabetic rats. These results indicate that inhibition of PDE3B by amrinone significantly augments insulinotropic action of physiological glucose in B-cells of normal rats. This effect is mediated via protein kinase A and may be also induced in the presence of metabolizable stimuli other than glucose. Effects generated by amrinone were demonstrated to be, however, insufficient to enhance glucose-induced insulin secretion in B-cells of streptozotocin-nicotinamide-induced diabetic rats.

The metabolic profile of growing lambs fed diets rich in unsaturated fatty acids.

The effect of two diets enriched with unsaturated fatty acids--one containing the addition of dried distillers grains with solubles (DGS) and the other the addition of false flax--Camelina sativa cake (CS)--on some metabolic parameters and hormone concentration in growing lambs was determined in this experiment. A total of 21 ram lambs of the Polish Whiteheaded mutton sheep were divided into three groups (the control, receiving DGS and CS). The diets were administered to animals for 6 weeks. During the experiment, peripheral blood was collected. Glucose (GL), total cholesterol (CH), triglycerides (TG), free fatty acids (FFA), insulin (IN), leptin (LE), triiodothyronine (T3) and thyroxine (T4) were assayed in serum. The age-dependent reduction in CH and TG limited by both experimental diets were observed. A significant increase in FFA concentration was observed in samples collected in the last, that is, third, time period. This was most probably caused by a 12-h pre-slaughter fasting period. A significantly lower dynamic of FFA increase in that experimental period was found in animals receiving the experimental feed. Insulin concentration in DGS-receiving lambs was increased, in contrast to the CS-receiving lambs, in which it was lower when compared to the control. LE concentration was decreased by both experimental diets, more markedly in the DGS-receiving animals. No differences between the experimental groups and the control were observed in T3 and T4 concentrations. The effect of 12-h pre-slaughter fasting was statistically highly significant for the levels of examined blood markers and hormones, except for TG and IN in the group of lambs receiving the experimental diet with CS.

Metabolic disturbances and defects in insulin secretion in rats with streptozotocin-nicotinamide-induced diabetes.

Rats with diabetes induced by streptozotocin (STZ) and nicotinamide (NA) are often used in animal studies concerning various aspects of diabetes. In this experimental model, the severity of diabetes is different depending on doses of STZ and NA. Moreover, diabetic changes in rats with STZ-NA-induced diabetes are not fully characterized. In our present study, metabolic changes and insulin secretion were investigated in rats with diabetes induced by administration of 60 mg of STZ and 90 mg of NA per kg body weight. Four to six weeks after diabetes induction, insulin, glucagon and some metabolic parameters were determined to evaluate the severity of diabetes. Moreover, insulin secretory capacity of pancreatic islets isolated from control and diabetic rats was compared. It was demonstrated that administration of 60 mg of STZ and 90 mg of NA per kg body weight induced relatively mild diabetes, since insulin, glucagon and other analyzed parameters were only slightly affected in diabetic rats compared with control animals. In vitro studies revealed that insulin secretory response was preserved in pancreatic islets of diabetic rats, however, was lower than in islets of control animals. This effect was observed in the presence of different stimuli. Insulin secretion induced by 6.7 and 16.7 mmol/l glucose was moderately reduced in islets of diabetic rats compared with control islets. In the presence of leucine with glutamine, insulin secretion appeared to be also decreased in islets of rats with STZ-NA-induced diabetes. Insulinotropic action of 6.7 mmol/l glucose with forskolin was also deteriorated in diabetic islets. Moreover, it was demonstrated that at a non-stimulatory glucose, pharmacological depolarization of plasma membrane with a concomitant activation of protein kinase C evoked significant rise in insulin release in islets of control and diabetic rats. However, in diabetic islets, this effect was attenuated. These results indicate that impairment in insulin secretion in pancreatic islets of rats with mild diabetes induced by STZ and NA results from both metabolic and nonmetabolic disturbances in these islets.

Effects of adenosine A(1) receptor antagonism on insulin secretion from rat pancreatic islets.

Adenosine is known to influence different kinds of cells, including beta-cells of the pancreas. However, the role of this nucleoside in the regulation of insulin secretion is not fully elucidated. In the present study, the effects of adenosine A(1) receptor antagonism on insulin secretion from isolated rat pancreatic islets were tested using DPCPX, a selective adenosine A(1) receptor antagonist. It was demonstrated that pancreatic islets stimulated with 6.7 and 16.7 mM glucose and exposed to DPCPX released significantly more insulin compared with islets incubated with glucose alone. The insulin-secretory response to glucose and low forskolin appeared to be substantially potentiated by DPCPX, but DPCPX was ineffective in the presence of glucose and high forskolin. Moreover, DPCPX failed to change insulin secretion stimulated by the combination of glucose and dibutyryl-cAMP, a non-hydrolysable cAMP analogue. Studies on pancreatic islets also revealed that the potentiating effect of DPCPX on glucose-induced insulin secretion was attenuated by H-89, a selective inhibitor of protein kinase A. It was also demonstrated that formazan formation, reflecting metabolic activity of cells, was enhanced in islets exposed to DPCPX. Moreover, DPCPX was found to increase islet cAMP content, whereas ATP was not significantly changed. These results indicate that adenosine A(1) receptor blockade in rat pancreatic islets potentiates insulin secretion induced by both physiological and supraphysiological glucose concentrations. This effect is proposed to be due to increased metabolic activity of cells and increased cAMP content.

Short-term regulation of adiponectin secretion in rat adipocytes.

Adiponectin belongs to the group of biologically active substances secreted by adipocytes and referred to as adipokines. Disturbances in its secretion and/or action are thought to be involved in the pathogenesis of some metabolic diseases. However, regulation of adiponectin secretion is poorly elucidated. In the present study, short-term regulation of adiponectin secretion in primary rat adipocytes was investigated. Isolated rat adipocytes were incubated in Krebs-Ringer buffer containing 5 mM glucose and insulin alone or in the combination with epinephrine, dibutyryl-cAMP, adenosine A(1) receptor antagonist (DPCPX), palmitate, 2-bromopalmitate or inhibitor of mitochondrial electron transport (rotenone). Adipocyte exposure for 2 h to insulin (1-100 nM) significantly increased secretion of adiponectin compared with secretion observed without insulin. Furthermore, secretion of adiponectin from adipocytes incubated with glucose and insulin was reduced by 1 and 2 microM epinephrine, but not by 0.25 and 0.5 microM epinephrine. Under similar conditions, 1 and 2 mM dibutyryl-cAMP substantially diminished secretion of adiponectin, whereas 0.5 mM dibutyryl-cAMP was ineffective. Secretion of adiponectin was found to be effectively decreased by DPCPX. Moreover, adipocyte exposure to rotenone also resulted in a substantial diminution of secretory response of adipocytes incubated for 2 h with glucose and insulin. It was also demonstrated that palmitate and 2-bromopalmitate (0.06-0.5 mM) failed to affect secretion of leptin. The obtained results indicated that in short-term regulation of adiponectin secretion, insulin and epinephrine exert the opposite effects. These effects appeared as early as after 2 h of exposure. Moreover, deprivation of energy or blockade of adenosine action substantially decreased secretion of adiponectin.

The inhibitory effect of resveratrol on leptin secretion from rat adipocytes.

BACKGROUND: Resveratrol was found to alleviate consequences of some metabolic disturbances which may be due to inappropriate dietary habits. It decreases mortality, increases insulin sensitivity and improves motor functions; these effects are accompanied by reduced plasma leptin and insulin. Leptin plays a significant role in the regulation of food intake and energy expenditure - elevated level in blood is one of the reasons of leptin-resistance and obesity. In this study, the direct effect of resveratrol on leptin secretion from isolated adipocytes was investigated. MATERIAL AND METHODS: Isolated rat adipocytes were incubated with resveratrol (62.5, 125 or 250 microM) and its effects on leptin secretion were studied. Cells were incubated with resveratrol in the presence of glucose (5 and 20 mM) and insulin (10 nM); glucose and nicotinic acid (1 mM); glucose and insulin in the presence of an inhibitor of protein kinase A (H-89, 50 microM) or alanine (10 mM) and insulin. The glucose uptake, glycerol release to the incubation medium, lactate and ATP produced by the cells were also measured. RESULTS: Resveratrol inhibited leptin secretion in all experimental designs in a dose-dependent manner. The effect was not accompanied by changes in glycerol release and glucose uptake. Adipocyte exposure to resveratrol enhanced the lactate formation. It was found that resveratrol dramatically reduced ATP in adipocytes. CONCLUSION: The obtained results revealed the direct ability of resveratrol to reduce leptin secretion from isolated rat adipocytes. Resveratrol is therefore a compound affecting the endocrine function of adipocytes.

Effects of adenosine A1 receptor antagonism on lipogenesis and lipolysis in isolated rat adipocytes.

Adenosine is secreted from adipocytes, binds to adenosine A(1) receptor and modulates various functions of these cells. In the present study, the effects of an adenosine A(1) receptor antagonist (DPCPX; 0.01, 0.1 and 1 microM) on lipogenesis, glucose transport, lipolysis and the antilipolytic action of insulin were tested in rat adipocytes. DPCPX had a very weak effect on lipogenesis and did not significantly affect glucose uptake. In adipocytes incubated with 1 microM DPCPX, lipolysis increased. This effect was blunted by insulin and by a direct inhibitor of protein kinase A. Moreover, 0.1 microM DPCPX substantially enhanced the lipolytic response to epinephrine and increased cAMP in adipocytes. However, DPCPX was ineffective when lipolysis was stimulated by direct activation of protein kinase A. Adipocyte exposure to epinephrine and insulin with or without 0.1 microM DPCPX demonstrated that this antagonist increased the release of glycerol. However, despite the presence of DPCPX, insulin was able to reduce lipolysis. It is concluded that DPCPX had a weak effect on lipogenesis, whereas lipolysis was significantly affected. The partial antagonism of adenosine A(1) receptor increased lipolysis in cells incubated with epinephrine alone and epinephrine with insulin due to the synergistic action of 0.1 microM DPCPX and epinephrine.

In vivo metabolic effects of naringenin in the ethanol consuming rat and the effect of naringenin on adipocytes in vitro.

Naringenin is a bioactive flavanone involved in the inhibition of drug metabolism which exhibits antioxidant, anti-inflammatory and anticancerogenic properties and which recently appeared to be a factor mitigating the hyperlipidaemic effects in rats and rabbits. In the performed experiment, the effect of naringenin, administered intragastrically (50 mg/kg) for 2 weeks to normal and ethanol drinking rats, on insulin and leptin levels and on some metabolic parameters was investigated. Naringenin did not change the hormone levels in any group of rats. Blood glucose, triglyceride, total, esterified and free cholesterol and high-density lipoprotein-cholesterol concentrations were also unaffected by this compound. Only free fatty acids were elevated after the naringenin treatment in the water-drinking rats. In spite of unchanged glucose and insulin concentrations in blood, the tested flavanone reduced the glucose/insulin ratio in ethanol-receiving rats. Liver triglycerides, elevated due to ethanol ingestion, were partially normalized by naringenin. Other tested parameters like liver glycogen and cholesterol, muscle triglycerides and glycogen were not altered in any group of rats. The influence of naringenin (62.5, 125, 250 and 500 microM) on basal and insulin-stimulated glucose conversion to lipids (lipogenesis) as well as on basal and epinephrine-stimulated glycerol release (lipolysis) in the isolated rat adipocytes was also tested. The basal and the stimulated lipogenesis tended to be decreased in the presence of the flavanone (250 microM). This inhibitory effect intensified and was statistically significant at the highest concentration of naringenin. The tested compound did not evoke any effect on basal lipolysis while the epinephrine-stimulated process was limited at the highest concentration of the flavanone. Naringenin (62.5, 125, 250 and 500 microM) had no effect on leptin secretion from the isolated rat adipocytes. Results obtained in our studies demonstrate that naringenin exerts a very weak influence on carbohydrate and lipid metabolism of normal and ethanol-consuming rats and on metabolism of isolated rat adipocytes.

Lack of the effect of mycotoxins-aflatoxin B1 and ochratoxin A on some functions of rat adipocytes.

Mycotoxins-aflatoxin B1 (AFB1) and ochratoxin A (OTA)-compounds which are strong carcinogenic, mutagenic and cytotoxic factors-are also known to evoke a decrease of food intake and body weight gains. The purpose of our study was to determine the direct influence of AFB1 and OTA incubated with isolated rat fat cells on the lipogenesis, lipolysis and leptin secretion. Adipocytes were isolated from the epididymal fat tissue by the collagenase digestion. Toxins used at concentrations 1, 10 and 100 microM were incubated for 90 min with adipocytes. Basal and insulin-stimulated lipogenesis-determined by the measure of [U-14C]glucose conversion to total lipids-was abated by AFB1 only at the highest concentration. At two lower ones, AFB1 did not affect the process. OTA at all used concentrations decreased insulin-stimulated lipogenesis but the effect was not dose-dependent. The lipolysis was determined by the measure of glycerol release from adipocytes. The basal lipolysis was unchanged by both toxins. The epinephrine-stimulated lipolysis was intensified by AFB1 only at the highest concentration, however, the process was not altered by OTA. The antilipolytic action of insulin was unaffected by both compounds (10 microM). To determine the influence of the tested toxins on leptin secretion, adipocytes were incubated for 120 min in the presence of glucose and insulin as stimulators of hormone secretion. AFB1 and OTA added to the incubation medium (1, 10 and 100 microM) had no significant influence on the leptin release. The results obtained in this experiment demonstrate that adipocytes are susceptible to the direct action of AFB1 and OTA. This susceptibility is, however, rather weak and is exhibited by a slight restriction of the lipogenesis (in the case of both toxins) and by a slight increase of the lipolysis (in the case of AFB1).

Leptin secretion and protein kinase A activity.

Leptin is an adipocyte-derived hormone participating in the regulation of food intake and energy balance. Its secretion from fat cells is potentiated by insulin and by substrates providing ATP, whereas factors increasing cAMP level attenuate hormone release stimulated by insulin and glucose. The present experiments were aimed to determine the effect of cAMP on leptin secretion stimulated by glucose, alanine or leucine in the presence of insulin. Moreover, the effect of protein kinase A inhibition on leptin secretion was tested. To stimulate leptin secretion, isolated rat adipocytes were incubated for 2 h in the buffer containing 5 mmol/l glucose, 10 mmol/l alanine or 10 mmol/l leucine, all in the presence of 10 nmol/l insulin. Inhibition of protein kinase A (PKA) by H-89 (50 micromol/l) slightly enhanced leptin release stimulated by glucose and leucine but not by alanine. Activation of this enzyme by dibutyryl-cAMP (1 mmol/l) substantially restricted leptin secretion stimulated by glucose, alanine and leucine. The inhibitory influence of dibutyryl-cAMP on leptin secretion was totally (in the case of stimulation induced by glucose) or partially (in the case of stimulation by alanine and leucine) suppressed by H-89. These results demonstrate that leptin secretion induced by glucose, alanine and leucine is profoundly attenuated by cAMP in PKA-dependent manner. Therefore, the action of different stimulators of leptin secretion may be restricted by agents increasing the cAMP content in adipocytes. Moreover, it has also been shown that inhibition of PKA evokes the opposite effect and enhances leptin release.

Short-term fasting and lipolytic activity in rat adipocytes.

The aim of this experiment was to study the influence of 18-hour food deprivation on basal and stimulated lipolysis in adipocytes obtained from young male Wistar rats. Fat cells from fed and fasted rats were isolated from the epididymal adipose tissue by collagenase digestion. Adipocytes were incubated in Krebs-Ringer buffer (pH 7.4, 37 degrees C) without agents affecting lipolysis and with different lipolytic stimulators (epinephrine, forskolin, dibutyryl-cAMP, theophylline, DPCPX, amrinone) or inhibitors (PIA, H-89, insulin). After 60 min of incubation, glycerol and, in some cases, also fatty acids released from adipocytes to the incubation medium were determined. Basal lipolysis was substantially potentiated in cells of fasted rats in comparison to adipocytes isolated from fed animals. The inhibition of protein kinase A activity by H-89 partially suppressed lipolysis in both groups of adipocytes, but did not eliminate this difference. The agonist of adenosine A (1) receptor also did not suppress fasting-enhanced basal lipolysis. The epinephrine-induced triglyceride breakdown was also enhanced by fasting. Similarly, the direct activation of adenylyl cyclase by forskolin or protein kinase A by dibutyryl-cAMP resulted in a higher lipolytic response in cells derived from fasted animals. These results indicate that the fasting-induced rise in lipolysis results predominantly from changes in the lipolytic cascade downstream from protein kinase A. The antagonism of the adenosine A (1) receptor and the inhibition of cAMP phosphodiesterase also induced lipolysis, which was potentiated by food deprivation. Moreover, the rise in basal and epinephrine-stimulated lipolysis in adipocytes of fasted rats was shown to be associated with a diminished non-esterified fatty acids/glycerol molar ratio. This effect was presumably due to increased re-esterification of triglyceride-derived fatty acids in cells of fasted rats. Comparing fed and fasted rats for the antilipolytic effect of insulin in adipocytes revealed that short-term food deprivation resulted in a substantial deterioration of the ability of insulin to suppress epinephrine-induced lipolysis.

The influence of fasting on liver sulfhydryl groups, glutathione peroxidase and glutathione-S-transferase activities in the rat.

Sulfhydryl groups, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) are important elements of the antioxidant defence in the organism. The efficacy of their antioxidant action is influenced by many factors. In this work, the effect of fasting on total, protein-bound and nonprotein sulfhydryl groups and on the activity of liver and serum GPx and GST in rats were determined. Male Wistar rats were divided into two groups: non-fasted and 18-hour fasted. In fasted animals liver content of nonprotein sulfhydryl groups (represented predominantly by reduced glutathione; GSH) was diminished by 22% in comparison to non-fasted group, whereas total and protein-bound -SH groups were unaffected. The activity of liver and serum GPx was unchanged in food deprived rats. In these animals the activity of GST in serum was reduced by 26%. Fasting had no significant effect on the activity of GST in the liver. Our results demonstrate that in rats deprived of food for 18 hours liver and serum GPx and GST are not involved in protection against action of reactive oxygen species formed during fasting. The observed drop in the content of liver nonprotein sulfhydryl groups without concomitant rise in the activity of GPx and GST indicates that this effect may be due to augmented degradation of GSH, its potentiated efflux from hepatocytes and formation of conjugates with intermediates arising as a result of reactive oxygen species action.

Adipocyte lipolysis, hormonal and metabolic changes in ethanol-drinking rats.

The effect of ethanol drinking on some hormonal and metabolic changes in the rat and on lipolysis in isolated adipocytes was tested. Male growing Wistar rats divided into two groups were used in the experiment. Ten percent ethanol solution as the only drinking fluid for 2 weeks depressed body weight gain. The diminution of blood insulin with simultaneous increase in leptin concentration found in these rats suggest that the physiological regulation of leptin secretion is disturbed by ethanol. Liver triglycerides content was substantially augmented due to ethanol ingestion. Adipocytes were isolated from both groups of rats by collagenase digestion and the lipolytic activity of these cells was compared. Isolated cells (10(6)/ml) were incubated for 90 min in Krebs-Ringer buffer (pH 7.4, 37 degrees C) containing 3 mm glucose and different lipolytic modulators: adrenaline (1 microm), insulin (1 nm), dibutyryl-cAMP (1 mm) and DPCPX (a selective antagonist of adenosine A1 receptor, 1 microM). To determine basal lipolysis cells were incubated without lipolytic agents. Lipolysis was determined by the amount of glycerol released from cells to the incubation medium. Basal and adrenaline-induced lipolysis was depressed in adipocytes of ethanol-drinking rats. The antilipolytic activity of insulin was the same in both groups of isolated cells. Lipolysis induced by dibutyryl-cAMP was only slightly reduced due to ethanol consumption, whereas triglycerides breakdown evoked by adenosine A1 receptor antagonism was unchanged. Results obtained in vitro indicate that subchronic ethanol drinking attenuates basal and stimulated lipolysis in adipocytes, however, the antilipolytic effect of insulin and the adenosine pathway are unchanged.

The effect of thyroid hormones on blood insulin level and metabolic parameters in diabetic rats.

The effect of exogenous thyroid hormones on blood insulin and metabolic parameters in diabetic rats was investigated. Three groups of rats were treated with streptozotocin (STZ; 50 mg/kg b.w., intravenously) and one group receiving only saline served as control. Beginning with the third day after STZ treatment, until the last day before decapitation, i.e. for 11 days, two groups of diabetic rats were treated with T3 (50 microg/kg b.w., i.p.) or T4 (250 microg/kg b.w., i.p.). After two weeks, STZ injected rats had lower body weight, hyperglycemia with a simultaneous drop in blood insulin and decrease of T3 and T4 concentrations in comparison to control animals. Liver glycogen content was also reduced, whereas serum lactate, free fatty acids, triglycerides and cholesterol were elevated. Exogenous thyroid hormones given to diabetic rats substantially attenuated hyperglycemia without any significant changes in blood insulin concentration. An additional reduction of body weight gain and depletion in liver glycogen stores were also observed. Thyroid hormones augmented serum lactate and cholesterol and had no beneficial effect on elevated free fatty acids and triglycerides. It can be concluded that in spite of partial restriction of hyperglycemia, thyroid hormones evoked several unfavourable changes strongly limiting their potential use in diabetes.

Streptozotocin induces lipolysis in rat adipocytes in vitro.

Streptozotocin (STZ) is used to induce experimental diabetes in animals and is also applied for the treatment of patients with insulinoma. The aim of the present work was to investigate the direct effect of STZ on lipolysis in isolated rat adipocytes. After the isolation, the cells were incubated in a Krebs-Ringer buffer of pH 7.4, at the temperature 37 degrees C for 90 min with different concentrations of STZ: 0.5, 1 or 2 mmol/l. STZ caused a significant rise in basal values (99%, 199%, and 377%, respectively) and epinephrine-stimulated (1 micromol/l) lipolysis (15%, 24% and 46%, respectively). Augmentation of basal lipolysis by STZ was neither restricted by insulin (1 nmol/l) nor by H-89 (an inhibitor of protein kinase A, 50 micromol/l). These results indicate the stimulatory influence of STZ on the action of hormone-sensitive lipase in isolated cells of white adipose tissue. The obtained outcomes suggest that in studies employing STZ, it is necessary to consider its direct effect upon lipolysis in adipocytes.