Frantisek Novak

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.

Postnatal development of phospholipids and their fatty acid profile in rat heart

The aim of this study was to determine the concentration of phospholipids (PL), plasmalogen components of choline (PC) and ethanolamine (PE) phosphoglycerides (PLPC, PLPE) and fatty acid profile of PL and triacylglycerols (TAG) in developing rat left ventricular myocardium between postnatal day (d) 2 and 100. The steepest increase of total PL (TPL) concentration occurs between d2 and d5, followed by a further slower increase between d20 and d40. Similar developmental changes were observed in PC and PE. The PLPE concentration rises by d10, whereas PLPC does not change during the whole period investigated, except for the transient decline on d5. The concentration of diphosphatidylglycerol (DPG) increases by d60; the steepest rise occurs between d20 and d40. Phosphatidylinositol (PI) concentration rises only by d5. The concentration of phosphatidylserine (PS) decreases between d5 and d10 and then it does not change. Sphingomyelin (SM) concentration is maintained till d10, it declines on d20 and does not change thereafter. The proportion of saturated fatty acids (SFA) increases by d5 in PC, PE, PS and TAG, and by d10 in DPG and PI. After d20 the SFA proportion gradually decline in all lipids. Monounsaturated FA (MUFA) proportion decreases in PC, PE, PI and PS from d2 till d10, and in the weaning period it tends to rise again. In contrast, in DPG and TAG the proportion of MUFA declines during the whole postnatal period. N-6 polyunsaturated FA (PUFA) decrease in all PL by d20 and rise again thereafter; in TAG they decline between d2 and d10 and return to the initial level by d100. N-3 PUFA increase in all PL during the suckling period and decline after weaning; in TAG they increase only by d5 and then they decline. This remodeling of myocardial PL and TAG composition during postnatal development may affect membrane properties and contribute to developmental changes in the function of membrane proteins and cell signaling.

Protein kinase C activity and isoform expression during early postnatal development of rat myocardium

Total protein kinase C (PKC) activity, its isoform expression, and concentration and fatty acid (FA) composition of diacylglycerol (DAG) were determined in the left ventricular myocardium of the rat during early postnatal development (d 2, 3, 5, 7, and 10). PKC activity measured by the incorporation of 32P into histone IIIS decreased between d 2 and 10 in the homogenate as well as in cytosolic, membrane (100,000g), and nuclear-cytoskeletal-myofilament fractions (1000g). Likewise, the expression of PKC isoforms (α, δ, and ε) determined by immunoblotting generally declined during the period analyzed, although with a variable pattern. In the membrane and nuclear cytoskeletal myofilament fractions, PKCδ and PKCε expression decreased markedly by d 3, returning to or close to the d 2 level immediately on d 5. PKCα expression in the membrane fraction remained almost unchanged by d 7, declining thereafter. PKCδ and PKCε were associated predominantly with particulate fractions, whereas PKCα was more abundant in the cytosolic fraction. DAG concentration exhibited a significant decline by d 5, consistent with the decrease in maximal PKC activity. The unsaturation index of FA in DAG tended to decrease on d 3 owing to the lowered proportion of all polyunsaturated FA of n−6 and n−3 series. These results demonstrate that the developmental decrease in PKC activity and expression in the rat myocardium is not linear and that subcellular localization of the enzyme exhibits isoform-specific day-by-day changes during the early postnatal period. These changes are compatible with the view that PKC signaling may be involved in the control of a rapid switch of myocardial growth pattern during the first week of life.

Aldosterone alters the phospholipid composition of rat colonocytes

Previous studies have shown that aldosterone treatment of amphibian epithelial cells results not only in stimulation of Na(+) absorption but also in changes in phospholipid composition which are necessary for the mineralocorticoid action of aldosterone. The present study was designed to investigate the effect of aldosterone on phospholipids of mammalian epithelia. Phospholipid and fatty acid composition was examined in colonic epithelium (mineralocorticoid target tissue) and thymus (non-mineralocorticoid but glucocorticoid target tissue) of rats which had received aldosterone or vehicle by a miniosmotic pump for 7 days. Aldosterone increased the mass of colonic phospholipids relative to cellular proteins with concomitant changes in the percentage distribution of fatty acids, whereas the relative distribution of membrane phospholipds was not changed. Phosphatidylcholine increased the content of polyunsaturated and decreased that of monounsaturated fatty acids, which predominantly reflected the accretion of arachidonic and a decrease in oleic and palmitoleic acids. Within the phosphatidylethanolamine subclass, pretreatment of rats with aldosterone decreased the content of monounsaturated fatty acids (predominantly oleic and palmitoleic acid) and of n-3 fatty acids, and increased the content of saturated fatty acids (palmitic acid). The saturated-to-nonsaturated fatty acid ratio also significantly increased after aldosterone treatment. No changes in thymic phospholipids were seen. The results are consistent with the contention that aldosterone specifically modulates phospholipid concentration and metabolism in mineralocorticoid target tissue. The changes in phospholipid content and its fatty acid composition during the fully developed effect of aldosterone may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.

Heterogeneity of insect flight muscle mitochondria as demonstrated by different phospholipid turnover in vivo

Abstract Homogenates of the flight muscle of Periplaneta americana were separated by differential centrifugation into several fractions. Examination by electron microscopy indicated that two of these fractions contain predominantly intact mitochondria. When expressed relative to the protein content they have a high content of diphosphatidylglycerol and their phospholipids incorporate [ 32 P]-orthophosphate in vivo much more slowly than phospholipids in the supernatant. The mitochondria sedimenting at the lower speed have, in vivo , a significantly lower incorporation or radioactive phosphate into diphosphatidylglycerol and phosphatidylethanolamine than those of the lighter fraction. From developing muscle, fractions of similar composition and yield can be obtained, but they do not differ in the rate of incorporation of radioactive phosphate into diphosphatidylglycerol in vivo .

Phospholipid metabolism in the flight muscle of Periplaneta americana during maturation

Abstract The incorporation rate of 32 P-orthophosphate into phospholipids of the metathoracic musculature of adult Periplaneta males decreases in the order: phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, sphingomyeline, and diphosphatidylglycerol. The kinetics of the incorporation into phosphatidylethanolamine strongly suggest that more than one pool is present. During the maturation process, which lasts in the muscle a little more than a week after adult ecdysis, the tissue permeability for phosphate is increased and the incorporation rate into diphosphatidylglycerol is more than 15 times higher than in one month old adults. There has been little change in the incorporation rate of the other phospholipids. It has been calculated that the formation of new mitochondrial membranes takes place without extensive breakdown of those already present in the tissue.

Phospholipid metabolism in red and white insect muscle

The red flight musculature of Schistocerca gregaria contains twice as much phospholipids than the white femoral musculature. In individual phospholipids the difference is greatest in phosphatidylethanolamine and phosphatidylglycerol, lowest in sphingomyeline and phosphatidylinositol. The plasmalogen content is very low. After an injection of 32P orthophosphate the increase of specific activity during six days follows a similar course in both muscle types in phosphatidylethanolamine, sphingomyeline and phosphatidylserine but is more rapid in red than in white muscle in phosphatidylcholine (1.3 ×) and in phosphatidylinositol (5 ×). The incorporation into diphosphatidylglycerol is extremely slow. Flight induces an increase in the specific activity in phosphatidylinositol.

Role of FAT/CD36 in novel PKC isoform activation in heart of spontaneously hypertensive rats.

Disruption to the sensitive balance of long-chain fatty acids and glucose in the heart could cause cardiovascular diseases. Searching for a possible role of novel protein kinase C (nPKC) in heart with disrupted energy balance, we compared the insulin-resistant spontaneously hypertensive rats (SHR), which carry a nonfunctional variant of the fatty acid transporter FAT/CD36, with the less insulin-resistant congenic strain SHR-4 that is genetically identical except for a segment on chromosome 4 including a wild-type gene for a functional FAT/CD36. We analyzed expression of the nPKC-δ and -ε isoforms plus triacylglycerols (TAG) content in the myocardium of both FAT/CD36 strains and after a high sucrose diet (HSD). Two weeks before killing, males of both strains were randomly divided into two groups and fed either a standard laboratory chow or an HSD. PKC was determined by Western blotting in particulate and cytosolic fractions from left ventricles. The SHR-4 rats exhibited lower serum levels of insulin and free fatty acids than did SHR rats and higher amounts of PKC-ε in the heart particulate fraction. HSD caused accumulation of heart TAG in SHR but not in SHR-4. HSD increased PKC-δ and decreased PKC-ε expression in particulate fraction from left ventricles of SHR-4 while having no effects in SHR. These results demonstrate that reduced insulin resistance in SHR-4 rats with wild-type FAT/CD36 is associated with the insulin signaling pathway involving nPKCs.

Increased phospholipid turnover in denervated insect muscle

Abstract 1. 1. Phospholipid metabolism was studied by [32P]-orthophosphate incorporation in vivo in unilaterally denervated metathoracic muscles of adult male cockroaches (Periplaneta americana) 14 days after denervation. 2. 2. No change in the wet weight or in the labelling rate of tissue inorganic phosphate and phosphoarginine was found. 3. 3. There was a slight decrease in the dry weight and phospholipid content in denervated muscle. 4. 4. There was an increase in the labelling rate of phospholipids in the denervated muscles ranging from 161% in phosphatidyl choline to 247% in diphosphatidyl glycerol. This increase was similar in three subcellular fractions examined.

Catabolism of uracil and cytosine in conventional and germ-free laboratory animals

Abstract 1. 1. The difference was studied between the catabolism of 2- 14 C-labelled uracil and cytosine in conventional and germ-free laboratory rodents. 2. 2. The rate of the uracil catabolism is the same in all the species investigated and is dependent neither on the way of application nor on the colonization of the intestine with the microflora. About 60% of the activity is expired as 14 CO 2 in 8 hr. 3. 3. After the peroral application, cytosine is catabolized to CO 2 most rapidly in guinea-pigs, and essentially more moderately in mice and rats. 4. 4. Mice and rats exhibit negligible catabolism of cytosine after the intraperitoneal application (up to 1.0%). 5. 5. Germ-free animals are catabolizing less than 0.5% of cytosine. 6. 6. In the urine of germ-free animals only non-metabolized cytosine was found, in conventional animals four further radioactive substances were present in addition to cytosine.