Inconsistent role of nitric oxide on lipolysis in isolated rat adipocytes

Though two isoforms of nitric oxide synthase, iNOS and eNOS, were reported in adipocytes, the role of NO in adipose tissue is still ambiguous. The aims of the present study were 1) to follow the effect of bacterial lipopolysaccharide (LPS), on 24 h-lipolysis in rat epididymal adipocyte culture in relation to iNOS stimulation; 2) to compare LPS-induced NO effects with exogenously NO, delivered as S-nitroso-N-acetylpenicillamine (SNAP), and 3) to examine the possible role of NO signaling agonist in lipolysis mediated by the beta(3)-adrenoreceptor agonist. Lipolysis was measured by glycerol and free fatty acid (FFA) production. The medium nitrite levels were used for the indirect estimation of NOS expression. Adipocyte mitochondrial function was assessed by the MTT test. LPS produced a concentration-dependent increase of NO with a decrease of viability at the highest dose. However, LPS did not affect lipolysis. SNAP did not exhibit significant changes in glycerol, FFA or MTT. BRL-37344 and db-cAMP significantly increased nitrite, glycerol and FFA levels. There was a positive correlation between glycerol release and nitrite production. Moreover, BRL-37344 significantly reduced mitochondrial functions. The pretreatment with bupranolol, beta(3)-antagonist, restored all parameters affected by BRL-37344. These results support a concept that NO fulfils multifaceted role of stimulating lipolysis under physiological conditions (beta-agonistic effect) and modulating the same processes during inflammatory (LPS) processes.     

Ontogenetic studies on adrenergic lipolysis in rat adipocytes from subcutaneous adipose tissue

The in vitro release of free fatty acids from small adipocytes (mean diameter 24.4 +/- 0.4 micrometer; 11-42 micrometer) isolated from subcutaneous adipose tissue of rats aged 5, 14, 21 and 35 days was induced by l-isoprenaline (ISO), l-noradrenaline (NA) or l-adrenaline (ADR). The results were related to a cell count of 10(6) and compared to results in adult rats [both sexes, fed ad libitum, mean weight 180-210 g). The cell yield decreased with development, i.e. it was highest in the youngest rats. In adult rats, increasing catecholamine (CA) concentrations produced pronounced concentration-dependent lipolysis in the potency order ISO greater than NA greater than ADR which is typical of beta 1-adrenergic functions. Qualitatively the same results in 5- and 35-day-old rats were observed, the latter showing greater sensitivity towards CA. Practically no lipolytic response was recorded in 14 and 21-day-old animals, however. Not one of the CA, nor any of the concentrations used, could evoke lipolysis in these animals. The experiments were carried out simultaneously on different age groups, technical errors can therefore be excluded.     

Deuterium oxide stimulates glucose metabolism and inhibits lipolysis in rat epididymal adipose tissue.

Incubation of segments of rat epididymal adipose tissue in media prepared with deuterium oxide results in increased glucose oxidation, increased lipogenesis, accelerated sugar transport and decreased lipolysis in response to epinephrine or theophylline. In view of the well documented action of heavy water to “stabilize” cytoplasmic microtubules, the foregoing observations are in support of a link between cytoplasmic microtubules and metabolic process in adipose tissue.     

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 (10⁶ cells/ml in plastic tubes at 37°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 estrification. 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 μM) and H-89 (an inhibitor of protein kinase A; 50 μM) 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 μM) lipolysis but failed to potentiate lipolysis induced by forskolin (1 μM) 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 phyestrogen 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.     

Beta-adrenergic agents increase the phosphorylation of phosphofructokinase in isolated rat epididymal white adipose tissue.

Pieces of rat epididymal adipose tissue were incubated in medium containing [32P]phosphate for 2 h to achieve steady-state labelling of intracellular phosphoproteins and then with or without hormones for a further 15 min. Phosphofructokinase was rapidly isolated from the tissue by use of either Blue Dextran-Sepharose chromatography or immunoprecipitation with antisera raised against phosphofructokinase purified from rat interscapular brown adipose tissue. Similar extents of incorporation of 32P into phosphofructokinase were measured by both techniques. Exposure of the tissue to adrenaline or the beta-agonist isoprenaline increased phosphorylation by about 5-fold (to about 1.4 mol of phosphate/mol of enzyme tetramer). No change in phosphorylation was detected with the alpha-agonist phenylephrine, but exposure to insulin resulted in an approx. 2-fold increase. The increased phosphorylation observed with isoprenaline was found to be associated with a decrease in the apparent Ka for fructose 2,6-bisphosphate similar to that observed on phosphorylation of phosphofructokinase purified from rat epididymal white adipose tissue with the catalytic subunit of cyclic AMP-dependent protein kinase. These results support the view [Sale & Denton (1985) Biochem. J. 232, 897-904] that an increase in cyclic AMP in adipose tissue may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.     

Effect of ketone bodies on lipolysis in adipose tissue in vitro

Norepinephrine-sensitive lipase activity was measured in rat epididymal fat pads by determining release either of free fatty acids or of glycerol. Stimulation of the lipase activity by norepinephrine in vitro could not be duplicated by injecting norepinephrine into the rats before sacrifice. A reliable method for assay of lipase deactivation rate was developed in which the tissue is incubated for 80 min, norepinephrine is added for a further incubation of 10 min, and the decay of lipase activity is measured during the next 10 min in the absence of hormone. Of the ketone bodies tested, -hydroxybutyrate and probably acetoacetate inhibited the activation of lipase by norepinephrine but had no effect on lipase deactivation rate, whereas acetone increased lipase activity stimulated by norepinephrine when tested at the concentration at which acetoacetate gave an inhibition. Substances other than -hydroxybutyrate that produce reduced nucleotides-alpha-glycerophosphate, malate, and ethanol-had no effect on lipase activity as tested in the present system.     

Calcium antagonists and lipolysis in isolated rat epididymal adipocytes: effects of tetracaine, manganese, cobaltous and lanthanum ions and D600.

The present study reports the effects on lipolysis occurring in isolated rat epididymal adipocytes of several agents which have each been found to interfere with membrane calcium transport in a variety of tissues. As reported by other workers, the local tetracaine was a strong inhibitor of hormone accelerated but not of basal lipolysis. The bivalent cations Mn2+ and Co2+ were similarly found to inhibit lipolysis stimulated with either epinephrine, ACTH, theophylline or dibutyryl cyclic AMP, whereas basal lipolysis was not markedly altered. This effect of Mn2+ and Co2+ was not mimicked by either Sr2+, Ba2+, Mg2+ or Ca2+. Cyclic AMP levels in adipocytes stimulated with epinephrine or ACTH tended to be higher in the presence of Mn2+ and Co2+. It is concluded, therefore, that Mn2+ and Co2+ inhibit lipolysis by uncoupling cyclic AMP accumulation from activation of triglyceride lipase. In contrast to Mn2+ and Co2+, the calcium antagonists La3+ and D600 were without effect on lipolysis. The antilipolytic effect of tetracaine, Mn2+ and Co2+ was found to persist in the absence of extracellular calcium, suggesting therefore that the antilipolytic effect of these drugs is unrelated to inhibition of calcium influx into adipocytes. The possibility is discussed that lipolytic agents cause an intracellular redistribution of calcium ion and that local anesthetics, Mn2+ and Co2+ interfere with lipolysis by preventing this intracellular redistribution of calcium.     

Conjugated linoleic acid supplementation caused reduction of perilipin1 and aberrant lipolysis in epididymal adipose tissue

MicroRNAs (miRNAs) have emerged as powerful regulators of multiple processes linked to human cancer, including cell apoptosis, proliferation and migration, suggesting that the regulation of miRNA function could play a critical role in cancer progression. Recent studies have found that human serum/plasma contains stably expressed miRNAs. If they prove indicative of disease states, miRNAs measured from peripheral blood samples may be a source for routine clinical detection of cancer. Our studies showed that both miR-508-3p and miR-509-3p were down-regulated in renal cancer tissues. The level of miR-508-3p but not miR-509-3p in renal cell carcinoma (RCC) patient plasma demonstrated significant differences from that in control plasma. In addition, the overexpression of miR-508-3p and miR-509-3p suppressed the proliferation of RCC cells (786-0), induced cell apoptosis and inhibited cell migration in vitro. Our data demonstrated that miR-508-3p and miR-509-3p played an important role as tumor suppressor genes during tumor formation and that they may serve as novel diagnostic markers for RCC.     

Calculation of lipolysis and esterification from glycerol metabolism in rat adipose tissue

Pieces of epididymal fat pad from fed and 48-hr-fasted rats were incubated for various periods of time in Krebs-Ringer bicarbonate containing [1-(14)C]glycerol. The radioactive substrate taken up by the tissue increased linearly with time in both groups and was mainly converted to glyceride-glycerol and CO(2). The slopes of the regressions of (14)C-labeled glyceride-glycerol with time were not different between the groups, while those of (14)CO(2) were smaller in the fasted than in the fed animals. Because the radioactive glycerol in the medium is being continuously diluted with the glycerol coming out of the tissue, it is necessary to take account of this factor in calculating the actual amount of glycerol utilized by the tissue. The glycerol produced by the tissues is higher in the fasted than in the fed animals, and in both groups it increases hyperbolically with time. As negligible amounts of the (14)C-labeled glycerol taken up by the tissue recirculates to the medium, the rates of glycerol release (lipolysis), esterification, and oxidation to CO(2) were calculated.     

Characterization of a colchicine receptor protein in rat epididymal adipose tissue

Colchicine was found to be taken up by adipose tissue and therein to bind to a soluble macromolecule not sedimented by centrifugation for 2 h at 100 000 × g. A similar binding occurred when soluble extracts of adipose tissue were incubated with colchicine. The binding reaction is temperature dependent and shows a pH optimum between 6.8 and 7.0. Double reciprocal plots of colchicine concentration versus amounts of colchicine bound to protein in the steady state disclosed an apparent Km of 0.250 to 1.5 ωM. The colchicine binding activity of soluble tissue extracts decreased when the extracts were incubated at 37°C. Addition of guanosine triphosphate and Mg²⁺ retarded the loss of colchicine binding activity. The molecular weight of the colchicine complex was estimated to be 115 000 and its sedimentation coefficient 5.8 S. All of these characteristics are remarkably similar to those of the protein tubulin which has been isolated from other tissues. Since it is now well known that tubulin is a protein subunit of cytoplasmic microtubules, it is suggested that the previously reported metabolic effects of colchicine on adipose tissue result from the dissolution of microtubules by colchicine.