Action of hydrocortisone on the lipid composition of active and inactive chromatin fractions in the rat liver

A composition of phospholipids and neutral lipids of rat liver chromatin and its active and inactive fractions has been investigated. It is shown that the hydrocortisone action results in marked increase in phospholipid/neutral lipid ratio of both chromatin and its active fraction. The changes in lipid content is clearly expressed in active chromatin fraction, the lipid content of inactive fraction is not changed. It is concluded that the increase of content in certain phospholipids and simultaneous decrease of neutral lipids in chromatin promotes the hormonal activation of genome.     

Shifts in phospholipid composition of the nuclear matrix in rat liver cells exposed to insulin

It was shown that the total content of phospholipids of rat liver nuclear matrix increased after in vivo action of insulin (2 units per 100 g of animal weight). At the same time the sphyngomyelin content was increased and the content of phosphatydilcholine and phoshpatydilinositol was decreased significantly under the hormone action. The decreasing of monophosphoinositides amount was accompanied by the increasing of triphosphoinositides content which indicates the redistribution among the phosphoinositides fractions under the hormone action and may be the result of insulin initiation of phosphoinositide pathway in nuclear membrances.     

Effect of hydrocortisone on composition of polyphosphoinositides in liver cell nuclear membrane and rat brain

The action of hydrocortisone in vivo was shown to cause changes both in the total amount of phospholipids and polyphosphoinositides of rat liver and brain nuclear membranes. The hormone increased the content of five from seven phospholipid fractions including the fraction of monophosphoinositide when acting in concentration and exposition leading to activation of biosynthetic processes. The increasing of monophosphoinositides amount was accompanied by the decreasing of triphosphoinositides content which indicated the redistribution among the phosphoinositides fractions under the steroid hormone action in both cell types.     

Synthesis of chromatin phospholipids

The presence of a phospholipid fraction associated with chromatin has been demonstrated by biochemical technique in rat hepatocytes. The composition of this fraction determined by chromatography with respect to that of the nuclei is characterized by low content of phosphatidylserine and high content in phosphatidylethanolamine. Also the synthesis and turnover studied after injection of [32P]O4(2-) show a different behaviour: the peak of activity is after 6 hrs in nuclei and microsomes, whereas in chromatin it occurs after 9 hrs. A second peak is evident after 24 hrs in chromatin and microsome phospholipids. Differences have been also shown by analyzing the single phospholipid radioactivity in time. The behaviour of chromatin phospholipids has also been studied during DNA premitotic synthesis in regenerating liver. It has been shown that there is no difference in synthesis in relation to that of DNA in nuclear phospholipids, whereas the specific activity of chromatin phospholipids begins to increase twelve hours after hepatectomy and continues throughout the period of the first mitotic wave, thus bringing to a summation with the beginning of the second wave. The role of this phospholipid fraction in relation to DNA synthesis and gene expression is discussed.     

Persistence of the effect of insulin on pyruvate dehydrogenase activity in rat white and brown adipose tissue during the preparation and subsequent incubation of mitochondria.

Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates. No effect on insulin was found if the hormone was added directly to mitochondria in the presence or absence of added plasma membranes. Concentrations of several possible regulators of pyruvate dehydrogenase kinase (ATP, ADP, NADH, NAD+, acetyl-CoA, CoA and potassium) were measured in rat epididymal-adipose-tissue mitochondria incubated under conditions where differences in pyruvate dehydrogenase activity persist as a result of insulin action. No alterations were found, and it is suggested that inhibition of the kinase is not the principal means by which insulin activates pyruvate dehydrogenase. The intramitochondrial concentration of magnesium was also unaffected. Differences in pyruvate dehydrogenase activity in interscapular brown adipose tissue associated with manipulation of plasma insulin concentrations of cold-adapted rats were also shown to persist during the preparation and subsequent incubation of mitochondria in the presence or absence of GDP. It is pointed out that the persistence of the effect of insulin on pyruvate dehydrogenase in incubated mitochondria will facilitate the recognition of the mechanism of this action of the hormone. Evidence that the short-term action of insulin involves an increase in pyruvate dehydrogenase phosphate phosphatase activity rather than inhibition of that of pyruvate dehydrogenase kinase is discussed.     

Changes in rat liver chromatin after administration of a mixture of amino acids

It was shown that injections of an amino acid mixture essentially increase the number of nuclease-sensitive regions of chromatin and its active fraction (Mg2+-soluble fraction), while hydrocortisone increases the amount of the latter, which is less sensitive to the effect of DNAase II. Genome activation during nonhormonal induction after administration of the amino acid mixture or during hydrocortisone injection reflects in different ways on the parameters of melting of chromatin active fractions and on the relative content of its protein fractions.     

Insulin effect on some biochemical and biophysical characteristics of lung surfactant

The incorporation of [14C]palmitic acid into rat alveolar wash total phospholipids and phospholipid fractions has been followed for 6, 8, 10 and 12 hr after insulin administration, indicating a considerable enhancement. The fatty acid profiles of phosphatidylcholines, phosphatidylethanolamines and phosphatidylglycerols were found changed after the hormone administration. Eight hours post insulin treatment the precursor incorporation was highest in all phospholipid fractions studied, as well as the contribution of long chain fatty acids. Dynamic monolayer studies of the lung wash lipid extracts indicated a maximally expanded lipid film corresponding to the highly unsaturated phospholipids present.     

Significance of serum for the preservation of insulin secretion during culture

The effects of serum and serum fractions on the maintenance of glucose-stimulated insulin secretion during culture of pancreatic islets were studied. The basal insulin release was independent of previous culture with serum but glucose-stimulated secretion increased with serum concentrations up to 0.3% which was sufficient for maximal effect. Although a high molecular size fraction (greater than 30,000 daltons) possessed the full activity of serum it is possible that the active principles are lighter compounds bound to serum proteins. Whereas the islet content of insulin was unaffected by culture with 3% serum, the presence of serum tended to increase the levels of pyridine nucleotides and the uptake of intracellular 45Ca in response to glucose. The sites of action for the serum factors are, therefore, the stimulus-secretion coupling and hormone discharge mechanisms rather than insulin biosynthesis.     

Effects of cortisol on lipid and lipoprotein synthesis in rat hepatocytes]

Under in vivo conditions cortisol induces moderate hyperlipidemia followed by an increase in the phospholipid and triglyceride concentrations in the blood and a decrease of cholesterol; similar changes were observed in the liver. At all time intervals studied cortisol inhibits the phospholipid and cholesterol syntheses and decreases the specific radioactivities of the lipids in the mitochondrial fraction. The hormone has an inhibiting effect on the fatty acid synthesis at early postinjection stages. The phospholipid synthesis is increased after adrenalectomy and is then inhibited after injection of the hormone. A single injection of ACTH or cortisol causes suppression of phospholipid and cholesterol syntheses and a decrease in their specific radioactivities in the mitochondria. A similar effect is observed under stress conditions. In addition, the hormone inhibits the synthesis of lipoprotein apoproteins of very low and high densities. After 5 hours following the hormone injection the lipoprotein apoprotein synthesis in the liver is activated; the activation of apoprotein synthesis is also observed after adrenalectomy. However, the injection of the hormone to adrenalectomized rats decreases the apoprotein synthesis. It was shown that in blood serum cortisol affects the conversions of very low density lipoproteins into low density lipoproteins, thus providing for hyperlipidemia.     

Changes induced by progesterone treatment in the sulphatides of the frog oviduct

Summary Sulphatides have been studied by histochemical and biochemical procedures in the oviduct of the frog in different experimental conditions. In ovariectomized or hypophysectomized, animals, compared to sham-operated, an increase in sulphatides was observed. The progesterone treatment did not significantly, modify this lipid fraction in ovariectomized frogs, while in hypophysectomized frogs it induced a further increase. Densitographic profiles of the sulphatides, obtained by thinlayer chromatography (TLC) and also recorded by Tesak equipment, were similar in ovariectomized or hypophysectomized frogs following hormone treatment because they showed three distinct fractions in both experimental groups of animals. The appearance of a third fraction never previously observed was probably induced by the progesterone treatment. Moreover, under the effects of this hormone, the phospholipid fractions (phosphatidylcholine and phosphatidylethanolamine) also showed different densitographic profiles.     

Selective time-dependent effects of insulin on brain phosphoinositide metabolism.

The effect of insulin on phosphoinositide metabolism in the cerebral cortex was examined using 32P as precursor. A maximal increase was detected as early as 15 s; phospholipid labeling declined after this initial peak but then increased to another maximum at 30 min. The levels of these phospholipids were unchanged at the earliest time examined, but at 30 min insulin caused an increase in the content of all phospholipids tested. In pulse-chase experiments, insulin stimulated depletion of 32P-labeled phosphoinositides only at 15 s. On the other hand, insulin treatment caused a biphasic diacyglycerol (DAG) production. We conclude that in cerebral cortex, insulin has a dual mechanism of action on phosphoinositide metabolism. First, insulin causes a rapid but transient hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism, followed by subsequent resynthesis; thereafter, insulin increases de novo phospholipid synthesis.     

The effect of low doses of external gamma irradiation on the chromatin structure and activity of the histone-specific proteinases of rat brain cells]

Irradiation of rats with doses of 0.5 to 2 Gy was shown to cause dose-dependent changes in the sensitivity of brain cell chromatin to the effect of DNAase I that were manifested by the increased level of DNA hydrolysis and a high content of the chromatin soluble fractions. The chromatin structure was only partially restored 24 h after irradiation. Changes in the chromatin structure were accompanied by the increase in the histone-specific proteinase activity.     

A phospho-oligosaccharide can reproduce the stimulatory effect of insulin on glycolytic flux in human fibroblasts

It has been recently demonstrated that insulin promotes the hydrolysis of a glycosyl-phosphatidylinositol, stimulating the release of a phospho-oligosaccharide which displays several insulin-like effects. In the present study we have investigated whether the compound is able to mimic insulin action on glucose metabolism in human fibroblasts. Similarly to the hormone, the phospho-oligosaccharide elicited a dose dependent increase in lactate output and fructose 2,6-bisphosphate content. The effect of the compound was time dependent with a progressive increase starting from 2 hours of incubation. 1 microM phospho-oligosaccharide had half maximal effect on both parameters, increasing glycolytic flux by approximately 30% and fructose 2,6-bisphosphate content by 70%. Therefore the phospho-oligosaccharide appears to be able to strictly reproduce insulin action on glucose metabolism in human fibroblasts.     

Effect of denervation on the phospholipid content in subcellular fractions of tonic and tetanic muscles.

Four weeks after denervation, various changes were observed in the phospholipid composition of the sarcolemmal and sarcoplasmic fractions of skeletal muscles with different functions. Neurotomy also affected the innervated contralateral muscles and produced opposite changes in the phospholipid content of subcellular fractions. The increase in the amount of phospholipids in the sarcolemmal fractions of the denervated muscles was only apparent. The difference between the denervated and contralateral muscles was also due to the decrease of phospholipids in the contralateral muscles. These changes were more pronounced in the tetanic (fast-twitch) than in the tonic (slow-twitch) muscles. In the sarcoplasmic fraction of the denervated tetanic muscle an increase, while in that of the tonic one a slight decrease of phospholipids appeared. In contrast, the phospholipid content in the sarcoplasmic fractions of contralateral muscles did not decrease, while it increased slightly in the tonic muscle. The amount of plasmalogens (fatty aldehyde: lipid phosphorus ratio) decreased only in the subcellular fractions of the denervated muscles while there was no change in those of the contralateral muscles.     

Effect of N-palmitoylethanolamine on phospholipid and fatty acid level in ischemic rat liver under reperfusion

The effect of 10(-5) M N-palmitoylethanolamine (NPE) on lipid peroxidation processes, phospholipid and fatty acid content in the ischemic rat liver under reperfusion was estimated. It was shown the significant accumulation of thiobarbituric acid reactive substances (TBARS), the increase of lysophosphatidylethanolamine (LPC), phosphatidylserine, phosphatidylethanolamine, sphingomyelin, general cholesterol content and also the decrease of phosphatidylcholine amount in the ischemic rat liver under reperfusion. Simultaneously the redistribution of saturated and unsaturated fatty acids quantity was detected, that indicates the increasing of membrane rigidity. The addition of NPE into the Eurocollins and Bretshneider's solutions under perfusion and preservation of a donor organ reduced the accumulation of TBARS, LPC, total cholesterol, prevented the changes of cholesterol/phospholipids and saturated/unsaturated fatty acids ratio value in the same experimental conditions. These effects, evidently, formed the basis of the protective action of NPE on the ischemic liver tissues under reperfusion.     

Insulin-mediated fusion of negatively charged phospholipid vesicles at low pH

Fusion of negatively charged phospholipid vesicles by bovine insulin was studied. The fusion induced by the hormone was demonstrated by resonance energy transfer, sepharose chromatography, light scattering and electron microscopy. The insulin effect was more effective when the pH was in the range of 3.6 - 3.9. The action of insulin also depends on the phosphatidylcholine: phosphatidic acid molar ratio, and buffer and vesicles concentration. At optimal conditions, half-maximal effect was obtained at 2 X 10(-8)M. The insulin-mediated fusion is non specific. The potential importance of these studies is discussed.     

Phospholipids in the nuclei and chromatin of the rat thymus at long periods following gamma-irradiation

The phospholipid content of the homogenate, nuclei and chromatin of rat thymus was being studied during three months after fractionated gamma-irradiation (2 Gy X 3 at a week interval). The number of phospholipids in the total fraction of phosphatidyl choline + phosphatidyl serine and phosphatidyl ethanolamine in the nuclei and chromatin of rat thymus was shown to decrease 60 min following the last exposure. In a month the phospholipid content in the nuclei and chromatin increased up to the control level keeping it throughout the entire period of observation.     

Diabetes affects phospholipid content in the nuclei of the rat liver.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on the content of different phospholipids and the incorporation of blood-borne 14C-palmitic acid into the phospholipid moieties in the rat liver nuclei. Diabetes was produced by intravenous administration of streptozotocin, and determinations were carried out two and seven days thereafter. Phospholipids were extracted from isolated nuclei and separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. Following that, they were quantified and radioactivity was measured. It was found that, in comparison to non-diabetic controls, two-day diabetes reduced the total content of phospholipids in the nuclei by 9.6%. The content of phospholipids in the nuclei by 9.6%. The content of phosphatidylcholine and phosphatidylserine was reduced and the content of the remaining phospholipids was stable. The specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin, based on radioactivity incorporated from 14C-palmitic acid, was elevated. Seven-day diabetes resulted in a reduction of the total phospholipid content in the nuclei by 39.4%. This was accounted for by a reduction in the content of each phospholipid fraction with the exception of cardiolipin. The specific activity of each phospholipid fraction, was elevated in this group. It is concluded that insulin is involved in the regulation of the nuclear phospholipid content.     

Nuclear maturation inducers in Bufo arenarum oocytes.

The present study analyses the effect of dihydrotestosterone (DHT) and mammalian insulin on the nuclear maturation of Bufo arenarum oocytes under in vitro conditions. The response of fully grown follicle oocytes to DHT, shown by germinal vesicle breakdown (GVBD), occurred in a manner dependent on dose, time and sexual cycle period. The highest oocyte sensitivity to the hormone appeared during the breeding period, a fact evinced by high GVBD percentages after short incubation periods and at a low hormone concentrations. Insulin also proved effective in inducing nuclear maturation, although its action was only visible at high concentrations and after a long incubation period. The combination of insulin and steroid hormones (DHT or progesterone), both at subliminal doses, caused a noticeable potentiating synergism, resulting in a rapid and important increase in GVBD. Another effect of insulin was the acquisition by oocytes of steroid sensitivity during folliculogenesis.