Acid sphingomyelinase inhibitors, imipramine and zoledronic acid, increase skeletal muscle tissue sensitivity to insulin action at old age.

Malfunction of skeletal muscles and dysregulated turnover of sphingolipids in the insulin responsive tissues have been determined at old age. Present article investigates the role of acid sphingomyelinase (SMase)-dependent ceramide accumulation in reduction of the skeletal muscle sensitivity to insulin action at old age. The 3-, 12- and 24-month-old Wistar male rats were used in the experiments. The progressive increase of ceramide content and ceramide/sphingomyeline (SM) ratio was determined in the extensor digitorum longus (EDL) muscle during aging of rats. The agedependent ceramide accumulation was followed by reduction of muscle tissue response to insulin action. The resistance of EDL to insulin action at old age can be imitated by exogenous natural N-palmitoyl-D-erythro-sphingosine (C16-ceramide) injection to adult rats, while imipramine or zoledronic acid treatment of old animals nullified dysregulation of SM turnover and improved the muscle tissue response to insulin action. Drugs significantly increased insulin-stimulated 2-D-[3H] glucose uptake by the EDL muscle of 24-month-old animals to the level close to that of 3-month-old rats in both in vivo and in vitro experiments. Imipramine, as well as zoledronic acid significantly reduced acid SMase activity in the EDL of old animals. Thus, ceramide overproduction via acid SMase activation can be important for the development of EDL resistance to insulin action. Therefore, acid SMase inhibitors can possibly be used as therapeutic tools for improvement of muscle tissue sensitivity to insulin action at an old age.

Role of acid sphingomyelinase in the age-dependent dysregulation of sphingolipids turnover in the tissues of rats.

Old age-associated pathologies usually coincide with altered sphingolipid metabolism. In the present article, the role of acid sphingomyelinase (ASMase) in the age-dependent changes of sphingomyelin (SM) and ceramide contents in the tissues has been investigated by means of ASMase inhibitors, imipramine and zoledronic acid. It has been determined that ceramide content and ceramide/SM ratio increased, while SM level decreased in the heart, liver, blood serum and skeletal muscles of 24-month old rats in contrast to 3-month old animals. Injections of imipramine or zoledronic acid to 24-month old rats resulted in significant downregulation of ASMase in the liver and skeletal and heart muscles. The both inhibitors decreased the ceramide content and ceramide/SM ratio and increased the SM content in all tissues studied, except the heart, of old rats to the levels close to those observed in the young animals. Long-term treatment of rats by inhibitors, which have different mechanisms of action on ASMase, exerts the similar, but not equal effects on enzyme activity and SM turnover. In summary, the data above strongly suggest that the age-dependent up-regulation of ASMase plays an important role in the modulation of ceramide and SM contents in rat tissues and that imipramine and zoledronic acid are useful tools for SM turnover manipulation at old age.

Modulation of Insulin Sensitivity of Hepatocytes by the Pharmacological Downregulation of Phospholipase D.

Background. The role of phospholipase D (PLD) as a positive modulator of glucose uptake activation by insulin in muscle and adipose cells has been demonstrated. The role of PLD in the regulation of glucose metabolism by insulin in the primary hepatocytes has been determined in this study. Methods. For this purpose, we studied effects of inhibitors of PLD on glucose uptake and glycogen synthesis stimulation by insulin. To determine the PLD activity, the method based on determination of products of transphosphatidylation reaction, phosphatidylethanol or phosphatidylbutanol, was used. Results. Inhibition of PLD by a general antagonist (1-butanol) or specific inhibitor, halopemide, or N-hexanoylsphingosine, or by cellular ceramides accumulated in doxorubicin-treated hepatocytes decreased insulin-stimulated glucose metabolism. Doxorubicin-induced hepatocytes resistance to insulin action could be abolished by inhibition of ceramide production. Halopemide could nullify this effect. Addition of propranolol, as well as inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) (wortmannin, LY294002) or suppressors of Akt phosphorylation/activity, luteolin-7-O-glucoside or apigenin-7-O-glucoside, to the culture media could block cell response to insulin action. Conclusion. PLD plays an important role in the insulin signaling in the hepatocytes. PLD is activated downstream of PI3-kinase and Akt and is highly sensitive to ceramide content in the liver cells.

Long-term food restriction prevents aging-associated sphingolipid turnover dysregulation in the brain.

Abnormalities of sphingolipid turnover in the brain during normal aging and age-related neurological disorders were associated with the neurons loss and cognitive malfunction. Calorie restriction (CR) prevented age-related deficits in hippocampal long-term potentiation and improved cognitive function at old age. In the paper we investigated the ceramide and sphingomyelin (SM) levels in the brain regions, which are critical for learning and memory of 3- and 24-month-old rats, as well as the correction of sphingolipid turnover in the brain of old rats, by means of the CR diet and modulators of SM turnover. Using the [methyl-(14)C-choline]SM, the neutral, but not the acid SMase activity has been observed to increase in both the hippocampus and brain cortex of 24-month-old rats with respect to 3-month-old animals. Age-dependent changes of neutral SMase activities were associated with ceramide accumulation and SM level drop in the brain structures studied. Treatment of the rats with the CR diet or N-acetylcysteine (NAC) or α-tocopherol acetate, but not an inhibitor of acid SMase imipramine, reduced the ceramide content and neutral SMase activity in the hippocampus of 24-month-old animals with respect to control rats of the same age. These results suggest that redox-sensitive neutral SMase plays important role in SM turnover dysregulation in both the hippocampus and neocortex at old age and that the CR diet can prevent the age-dependent accumulation of ceramide mainly via neutral SMase targeting.

Vitamin E prevents the age-dependent and palmitate-induced disturbances of sphingolipid turnover in liver cells.

Sphingolipid turnover has been shown to be activated at old age and in response to various stress stimuli including oxidative stress. Reduction of vitamin E content in the liver under the pro-oxidant action is associated with enhanced sphingolipid turnover and ceramide accumulation in hepatocytes. In the present paper, the correction of sphingolipid metabolism in the liver cells of old rats and in the palmitate-treated young hepatocytes using α-tocopherol has been investigated. 3- and 24-month-old rats, [(14) C]palmitic acid, [methyl-(14) C-choline]sphingomyelin (SM), and [(14) C]serine were used. α-Tocopherol administration to old rats or addition to the culture medium of old liver slices or hepatocytes prevented age-dependent increase of ceramide synthesis and lipid accumulation, and increased SM content in liver tissue and cells. α-Tocopherol treatment of old cells decreased the neutral and acid sphingomyelinase (SMase) activities in hepatocytes and serine palmitoyl transferase activity in the liver cell microsomes. Effect of α- or γ-tocopherol, but not of δ-tocopherol, on the newly synthesized ceramide content in old cells was correlated with the action of inhibitor of serine palmitoyl transferase (SPT) activity (myriocin) and SMase inhibitors (glutathione, imipramine). Addition of α-tocopherol as well as myriocin to the culture medium of young hepatocytes, treated by palmitate, abolished ceramide accumulation and synthesis. The data obtained demonstrate that α-tocopherol normalized elevated ceramide content in the old liver cells via inhibition of acid and neutral SMase activities and lipid synthesis de novo. α-Tocopherol, reducing ceramide synthesis, prevented palmitate-induced aging-like ceramide accumulation in young liver cells.

Effects of long-term fish oil-enriched diet on the sphingolipid metabolism in brain of old rats.

An increase of the neutral sphingomyelinase (SMase) activity in the animal brain at old age is associated with a higher expression of the inflammatory marker, interleukin-1beta and a cognitive decline. Dietary n-3 polyunsaturated fatty acids (n-3 PUFA) are found to exhibit anti-inflammatory effects and normalize the age- or dementia-related cognitive function decline. In the paper we investigate the ceramide, sphingomyelin (SM) and phosphatidylserine (PS) levels in the hippocampus of 3-month-old-32-month-old rats as well as the correction of sphingolipid turnover in the brain of old rats by means of the dietetic fish oil and PS. To study the lipid turnover, the [(14)C]-labeled SM and palmitic acid were used. The [(14)C]ceramide level significantly increased while those of the [(14)C]SM, SM and PS levels decreased in the hippocampus of aged rats as compared with 3-month-old animals. Treatment of the rats with the fish oil or PS reduced the [(14)C]ceramide content and neutral SMase activity in the hippocampus of the 24-month-old animals with respect to control rats of the same age. These results suggest that dietary n-3 PUFA can prevent the age-dependent abnormalities of sphingolipid turnover via increased PS content in the hippocampus of old rats.

Effects of flavonoids on sphingolipid turnover in the toxin-damaged liver and liver cells.

BACKGROUND: The ceramide generation is an early event in the apoptotic response to numerous stimuli including the oxidative stress and ceramide analogs mimic the stress effect and induce apoptosis. Flavonoids of German chamomile are reported to exhibit the hepatoprotective effect. Flavonoids affect sphingolipid metabolism and reduce the elevated ceramide level in the aged liver. In the present paper, the ceramide content and production in the CCl4- and ethanol-treated liver and hepatocytes as well as the correction of sphingolipid metabolism in the damaged liver using the mixture of German chamomile flavonoids (chamiloflan) or apigenin-7-glucoside (AP7Glu) have been investigated. RESULTS: The experiments were performed in either the rat liver or hepatocytes of normal, CCl4- and ethanol-treated or flavonoid- and toxin plus flavonoid-treated animals. [14C]palmitic acid and [methyl-14C-phosphorylcholine]sphingomyelin were used to investigate the sphingolipid turnover. Addition of the CCl4 or ethanol to isolated hepatocyte suspensions caused loss of cell viability and increased the lactate dehydrogenase release from the cells into supernatant and ceramide level in the cells. CCl4 administration to the rats enlarged ceramide mass as well as neutral sphingomyelinase (SMase) activity and reduced ceramide degradation by the neutral ceramidase. Pretreatment of isolated hepatocytes with flavonoids abrogated the CCl4 effects on the cell membrane integrity and normalized the ceramide content. Flavonoid administration to the rats normalized the elevated ceramide content in the damaged liver via neutral SMase inhibition and ceramidase activation. CONCLUSION: The data obtained have demonstrated that flavonoids affect sphingolipid metabolism in the CCl4- and ethanol-damaged liver and liver cells. Flavonoids normalized activities of key enzymes of sphingolipid turnover (neutral SMase and ceramidase) and ceramide contents in the damaged liver and liver cells, and stabilized the hepatocyte membranes.

Effects of Chamomilla recutita flavonoids on age-related liver sphingolipid turnover in rats.

The increased sphingolipid turnover in the liver is associated with elevation of free radical production and state of chronic inflammation at old age. Plant polyphenols are reported to exhibit antioxidant and anti-inflammatory effects. In the present paper, the lipids contents and ceramide production in the liver and hepatocytes as well as the correction of sphingolipid metabolism at old age using the mixture of Chamomilla recutita flavonoids (chamiloflan) or apigenin-7-glucoside or luteolin-7-glucoside alone have been investigated. To study the sphingolipids turnover, the [14C]serine-pre-labeled hepatocytes and [14C-methyl]- or [14C]palmitate-pre-labeled sphingomyelin (SM) and ceramide were used. The ceramide content was higher in the liver and hepatocytes of 24- and 27-28-month-old animals as compared to adult 3-month-old Wistar rats. An addition of flavonoids to the culture medium did not influence significantly on the lipids contents and metabolism in the isolated hepatocytes. The administration of flavonoids to old rats decreased the elevated neutral and acid SMases activities and ceramide mass and did not affect both the lipid content in the liver of adult animals and ceramide conversion to the sphingosine or SM. These results suggest that the SMases play a key role in the flavonoid-induced decrease of ceramide levels in the liver of old rats.

Long- and short-term effects of thyroxine on sphingolipid metabolism in rat liver.

BACKGROUND: Thyroid hormones are well known as modulators of signal transduction. Early experiments showed that sustained elevation of diacylglycerol (DAG) and ceramide levels in liver cells of hypothyroid rats is an essential prerequisite for disturbances in agonist responsiveness of hepatocytes. In this paper the effects of thyroxine (L-T(4)) on sphingolipid mass, synthesis, and degradation are investigated. MATERIAL/METHODS: The experiments were performed on either [1(14)C]CH(3)COOH-labeled rat liver or hepatocytes and isolated liver cell plasma membranes. To determine sphingolipid synthesis and degradation in the liver, [(14)C]palmitic acid, [methyl-(14)C]-labeled sphingomyelin (SM), and phosphatidylcholine were used. RESULTS: The administration of L-T(4) to animals was accompanied by a time-dependent accumulation of newly synthesized ceramide, glucosyl ceramide, and SM in liver. In hyperthyroid liver, prominent ceramide accumulation and acidic SMase activation was found. The addition of hormone in a physiological dose to the incubation or perfusion medium led to a rapid stimulation of de novo ceramide synthesis, transient lipid accumulation in the liver, followed by SM synthase activation. CONCLUSIONS: Our results indicate that SMases, ceramide synthesis de novo, and SM synthase are under thyroid hormone control. The level of ceramide is markedly elevated in the hyperthyroid liver via the activation of acidic SMase and lipid synthesis de novo. In experiments in vitro, the hormone induces a short-lived accumulation of sphingolipid, probably due to its reduced efflux from the liver.

Effects of thyroxine and 1-methyl, 2-mercaptoimidazol on phosphoinositides synthesis in rat liver.

BACKGROUND: Phosphoinositides mediate one of the intracellular signal transduction pathways and produce a class of second messengers that are involved in the action of hormones and neurotransmitters on target cells. Thyroid hormones are well known regulators of lipid metabolism and modulators of signal transduction in cells. However, little is known about phosphoinositides cycle regulation by thyroid hormones. The present paper deals with phosphoinositides synthesis de novo and acylation in liver at different thyroid status of rats. RESULTS: The experiments were performed in either the rat liver or hepatocytes of 90- and 720-day-old rats. Myo-[3H]inositol, [14C]CH3COONa, [14C]oleic and [3H]arachidonic acids were used to investigate the phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis. 1-methyl, 2-mercaptoimidazol-induced hypothyroidism was associated with the decrease of myo-[3H]inositol and [3H]arachidonic acids incorporation into liver phosphoinositides and total phospholipids, respectively. The thyroxine (L-T4) injection to hypothyroid animals increased the hormones contents in blood serum and PtdInsP2 synthesis de novo as well as [3H]arachidonic acids incorporation into the PtdIns and PtdInsP2. Under the hormone action, the [14C]oleic acid incorporation into PtdIns reduced in the liver of hypothyroid animals. A single injection of L-T4 to the euthyroid [14C]CH3COONa-pre-treated animals or addition of the hormone to a culture medium of hepatocytes was accompanied by the rapid prominent increase in the levels of the newly synthesized PtdIns and PtdInsP2 and in the mass of phosphatidic acid in the liver or the cells. CONCLUSIONS: The data obtained have demonstrated that thyroid hormones are of vital importance in the regulation of arachidonate-containing phosphoinositides metabolism in the liver. The drug-induced malfunction of thyroid gland noticeably changed the phosphoinositides synthesis de novo. The L-T4 injection to the animals was followed by the time-dependent increase of polyphosphoinositide synthesis in the liver. The both long-term and short-term hormone effects on the newly synthesized PtdInsP2 have been determined.

Inhibition of protein tyrosine kinase activity disrupts early retinal development.

In the present study, we have investigated the role of tyrosine kinase activity during early retinal development in Xenopus laevis. The protein tyrosine kinase (PTK) inhibitors lavendustin A and genistein were used to determine the possible role of tyrosine kinase activity during retinal development in vivo and in vitro. Application of the inhibitors to early embryonic retina disrupted the pattern of lamination in the developing retina. The plexiform layers were severely disorganized or were no longer apparent, and photoreceptor morphogenesis was disrupted. Immunocytochemical analysis verified the presence of focal adhesions in dissociated retinal neuroepithelial cells isolated from St 25 embryos. Application of the PTK inhibitors blocked focal adhesion assembly in these primary cultured cells. To further investigate the regulation of focal adhesions by PTK activity, we examined the effect of lavendustin A on cultured XR1 glial cells. Lavendustin A produced a dose-dependent decrease in the proportion of XR1 cells displaying focal adhesions. Taken together, these results suggest that tyrosine kinase activity is essential for regulating neuroepithelial cell adhesion, migration and morphogenesis during retinal development. Furthermore, the disruption of retinal development may, in part, be due to the inhibition of integrin-mediated signaling.

Increase in diacylglycerol production by liver and liver cell nuclei at old age.

Early experiments have shown that sustained elevation of diacylglycerol (DAG) level in liver cell plasma membranes of aged rats is an essential prerequisite for disturbances in agonist responsiveness of hepatocytes. However, the mechanisms responsible for the age-related rise in DAG content in liver cells have not been clearly identified. The aim of the present study was to determine if phospholipid degradation by endogenous phospholipases precedes the DAG accumulation in liver cells and cell nuclei. The DAG formation in liver slices, hepatocytes and liver cell nuclei has been studied in 90-and 720-day-old rats. The experiments were performed in either the [14C]CH(3)COOH-labeled rat liver or liver slices, or hepatocytes pre-labeled by [14C] linoleic or [14C] oleic or/and [3H] arachidonic acid. The metabolism of [14C-linoleil]phosphatidylcholine (PC) and [14C-methyl] PC was investigated in isolated liver cell nuclei. The [14C]DAG production in pre-labeled liver slices and in hepatocytes increased significantly and [14C]phosphatidylethanol formation decreased with age. DAG formed in liver of old animals showed labeling ratios (14C/3H) close to those of PC, pointing to PC as the primary DAG source in senescent liver. The liver slices of old rats, pre-labeled in situ by [14C]CH(3)COOH, demonstrated the enhanced ability to produce DAG and sphingomyelin (SM) in a time-dependent manner while the PC level decreased in liver of old rats. The production of [14C]DAG, [14C]SM and [14C]phosphocholine in the nuclei of old rats was significantly higher than that in adult animals. These results suggest that PLC and SM synthase activities play a key role in a regulation of DAG basal levels present in the liver cells and in the nuclei of old rats.

Drug-induced and postnatal hypothyroidism impairs the accumulation of diacylglycerol in liver and liver cell plasma membranes.

BACKGROUND: Thyroid hormones are well known modulators of signal transduction. The effect of hyper- and hypo-thyroidism on diacylglycerol/protein kinase C (DAG/PKC) signaling in cardiomiocytes has been determined. Triiodothyronine (T3) has been shown to prevent the alpha1-adrenoreceptor-mediated activation of PKC but does not alter the stimulation of enzyme and hepatic metabolism by phorbol ethers. It has been suggested that the elevation of endogenous DAG in senescent or hypothyroid cells changes the PKC-dependent response of cells to phorbol esters and hormones. In the present study, was examined the formation of DAG and activation of PKC in liver cells from rats of different thyroid status. RESULTS: The results obtained provide the first demonstration of DAG accumulation in liver and cell plasma membranes at age- and drug-dependent thyroid gland malfunction. The experiments were performed in either the [14C]CH3COOH-labeled rat liver, liver slices or hepatocytes labeled by [14C] oleic acid and [3H]arachidonic acid or [14C]palmitic acid as well as in the isolated liver cell plasma membranes of 90- and 720-day-old rats of different thyroid status. The decrease of T4 and T3 levels in blood serum of 720-day-old rats and mercazolil-treated animals was associated with increases of both the DAG mass in liver and liver cell plasma membranes and newly synthesized [14C]DAG level in liver and isolated hepatocytes. Hypothyroidism decreased PKC activity in both membrane and cytosol as well as phospholipid and triacylglycerol synthesis in liver. These hypothyroidism effects were restored in liver by injection of T4. T4 administration to the intact animals of different ages decreased the DAG level in liver and isolated plasma membranes and the content of newly synthesized DAG in liver. The reduction of DAG level in liver was not associated with increasing free fatty acid level. DAG labeling ratio 14C/3H in liver slices of rats of different thyroid state sharply differed from PL. DAG was relatively enriched in [14C]oleic acid whereas PL were enriched in [3H]arachidonic acid. CONCLUSIONS: The above data have indicated that thyroid hormones are important physiological modulators of DAG level in rat liver and cell plasma membranes. Age- and drug-induced malfunction of thyroid gland resulted in a prominent decrease of glycerolipid synthesis which may promote DAG accumulation in liver.