Ilaria Parentini

Ageing and oxidative stress: A role for dolichol in the antioxidant machinery of cell membranes?

Dolichol is a polyprenol compound broadly distributed in membranes, biosynthetized by the general isoprenoid pathway from acetate via mevalonate and farnesyl pyrophosphate. Dolichol lays inside the membrane between the two leaflets of the lipid bilayer very close to the tail of phospholipid fatty acids. No definite catabolic pathways for this molecule have yet been identified. Evidence is produced that dolichol levels increase dramatically with increasing age; that anti-ageing caloric restriction retards this age-associated change; that dolichol may act as a radical scavenger of peroxidized lipids belonging to the cell membranes. In view of the polyunsaturated fatty acids (PUFA), dolichol and Vitamin E location and stechiometry, it is proposed that molecules might interact each-other to form a highly matched free-radical-transfer chain, whose malfunctioning might be involved in statin toxicity and neurodegenerative diseases.

Anti-aging effects of anti-lipolytic drugs.

Genetic disruption of insulin and insulin-like signaling pathways may extend lifespan. Hyperinsulinemia and insulin resistance may accelerate aging. The hypothesis was tested that a once-a-week life-long inhibition of insulin secretion by the administration of anti-lipolytic drugs might have anti-aging effects. Groups of 3-month-old male Sprague-Dawley rats were (a) given standard laboratory food ad libitum (AL); (b) fed AL 6 days and fasted 1 day every week (FW); (c) fed AL every other day (EOD), (d) fed like FW and given Acipimox (50 mg/kg b.w.) on the day of fasting (FWA) by the gastric tube. The AL, FW and EOD groups received saline intragastrically. Treatment with ACIPIMOX transiently decreased plasma free fatty acids, glucose and insulin and increased valine plasma levels, and had no long-term effect on food consumption and body weight. By age 6, 12 and 24 months subgroups were taken and the age-related changes in liver dolichol and autophagic proteolysis--which are correlated with life-expectancy--were measured. Liver dolichol levels increased and autophagic proteolysis decreased in mature and older AL rats; EOD and FWA fully counteracted these changes; FW rats had significant but smaller beneficial effects. It is concluded that life-long weekly-repeated transient inhibition of insulin secretion by antilipolytic drugs may have an anti-aging effect, additive to the anti-aging effect of a milder caloric restriction. Speculation is that transiently lower plasma insulin levels might stimulate the anti-aging cell-repair mechanism autophagy, which has longer lasting effects on cell housekeeping.

The age-related accumulation of dolichol in rat liver may correlate with expectation of life

In order to test the hypothesis that the ageing-related alteration in membrane lipids might reflect the biological age of rodents, the levels of liver dolichol were assayed by the HPLC procedure in male ad-libitum fed (AL) Sprague-Dawley rats aged 2, 6, 12 and 24months, and in 24-month-old rats on anti-aging food-restrictions (FR) differing in duration and in their effects on longevity. Results showed that the effects on liver dolichol of FR initiated at 2, 6 and 12 months of age, or initiated at 2 and interrupted at 18 months of age were significantly different, and reflected the differences in the effects of FR on expectation of life (the longer the expected residual lifespan the lower the content in liver dolichol). The conclusion is that assay of the quantity of dolichol in the liver tissue may be used as a marker of the biological age of the animal and therefore as an important biomarker of ageing.

The effect of carbon tetrachloride and ultraviolet radiation on dolichol levels in liver cells isolated from 3- and 24-month-old male Sprague-Dawley rats

Dolichol (D) is a long-chain polyprenoid broadly distributed in the cell membranes, possibly endowed with a free-radical scavenging activity, whose concentration in tissues increases with increasing age. No enzyme pathway for D degradation has been discovered. In order to test the hypothesis that D might undergo a non-enzymatic free-radical mediated decomposition the effects of a xenobiotic agent (carbon tetrachloride, CCl4) and ultraviolet-B (UV-B) radiation on D levels were studied in liver cells isolated from male ad libitum fed Sprague-Dawley rats aged 3 or 24 months. Liver cells (90 mg/ml) were incubated in sealed flasks (6 ml cell suspension each) for 0, 5, 10and 20 min after the addition of 25, 50 or 200μl CCl4 in the central well. 50 ml of a 6 mg/ml liver cell suspension were poured in a 120 cm2 Petri dish and the sediment liver cell monolayer was exposed to UV Bradiation for 0, 5, 10, 20 and 40 min. At the given time, cells were taken and D was extracted and assayed by the HPLC procedure. D levels were remarkably higher in older than in younger cells as expected (P < 0.001). Treatment with CCl4 and UVB caused a highly significant decrease in D (P < 0.001) whose percentage was larger in younger than in older cells. The conclusions are that free-radicals generated either by chemical or by physical agents cause a very rapid depletion of D in liver cells, and that the effect of the free radical attack on D decomposition may be lower percentage wise in older than in younger cells, which might account at least in part for the accumulation of D in older tissues.

Effect of increasing age on tissue dolichol levels in ad libitum fed and food-restricted rats

In order to test the hypothesis that the ageing-related alteration in membrane lipids might reflect the biological age of rodents, we studied the effects of age in ad libitum fed (AL)and food-restricted (FR) male Sprague-Dawley rats on the levels of dolichol in different organs involved [liver (L) and kidney (K)] or not involved [brain (B), sciatic nerve (SN),heart (H), soleus (S) and extensor digitorum longus (EDL) muscles] in dolichol excretion. At the given age, tissue dolichol was extracted and assayed by HPLC procedure. Results show that the levels of dolichol were significantly different in different tissues and increased dramatically with increasing age. The anti-ageing FR regimen had significant preventive effects on dolichol accumulation in the excretory organs. The effect of FR on the liver was much bigger than that of kidney. The effect of FR retarding dolichol accumulation in the liver co-varied with the effects of FR on longevity. In conclusion, these data show that the quantity of dolichol in the hepatic tissue might be used as a marker of the biological age of the animal.

Dolichol levels in younger and older rat hearts heterotopically transplanted in younger recipients

Dolichol (D) levels increase dramatically in older tissue. An understanding of the exchangeability of D between tissues may be essential in order to understand the mechanism of the abnormal accumulation associated with aging. The question was investigated by the use of organ transplantation. D-poor hearts donated by 3-mon-old and D-rich by 22-mon-old male Lewis rats were transplanted heterotopically in 3-mon-old syngenic recipients, whose peripheral tissues and liver were poor in D. Native and transplanted hearts were taken 7 and 21 d after surgery. Native hearts of 3-mon- and 22-mon-old male Lewis rats served as control. D concentration and quantity were higher in older than in younger native hearts as expected. In the transplanted hearts, the quantity of D was unchanged, irrespective of the age of the donor and of the time of transplantation, whereas D concentration increased because of the remarkable disuse atrophy. No changes in D were observed in recipients’ tissues. It is concluded that dolichol is not redistributed via circulation from the transplanted heart to the tissues and liver of the younger recipient.

The fate of dolichol in rat cells and tissues.

Dolichol (D) levels increase dramatically in older tissue. A better understanding of the fate of cell D and exchange between tissues could be essential for understanding the mechanism of the abnormal accumulation. The fate of red blood cell D was investigated by the use of phenylhydrazine-induced hyperhaemolysis. The effect of atrophy on D tissue levels was studied in the perineal muscles of castrated rats. Influence of D transportation between tissues on the levels of D was studied by the use of age-mismatched heterotopic transplantation of D-rich-hearts from older (22 months old) donor rats in younger (3 months old) D-poor syngenic recipients. Increased red blood cell destruction by splenic macrophages did not cause accumulation but rather a significant depletion of the D content of the spleen. The shrinkage of tissues by endocrine or disuse atrophy did not affect the D content of muscle, where D concentration increased. No significant net redistribution of D was observed from the transplanted older heart to liver and tissues of younger recipients. In conclusion, phagocytosis appears to be the only process resulting in the disposal of tissue D.

Dolichol: A Component of the Cellular Antioxidant Machinery

Dolichol, an end product of the mevalonate pathway, has been proposed as a biomarker of aging, but its biological role, not to mention its catabolism, has not been fully understood. UV-B radiation was used to induce oxidative stress in isolated rat hepatocytes by the collagenase method. Effects on dolichol, phospholipid-bound polyunsaturated fatty acids (PL-PUFA) and known lipid soluble antioxidants [coenzyme Q (CoQ) and α-tocopherol] were studied. The increase in oxidative stress was detected by a probe sensitive to reactive oxygen species (ROS). Peroxidation of lipids was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). Dolichol, CoQ, and α-tocopherol were assessed by high-pressure liquid chromatography (HPLC), PL-PUFA by gas–liquid chromatography (GC). UV-B radiation caused an immediate increase in ROS as well as lipid peroxidation and a simultaneous decrease in the levels of dolichol and lipid soluble antioxidants. Decrease in dolichol paralleled changes in CoQ levels and was smaller to that in α-tocopherol. The addition of mevinolin, a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR), magnified the loss of dolichol and was associated with an increase in TBARS production. Changes in PL-PUFA were minor. These findings highlight that oxidative stress has very early and similar effects on dolichol and lipid soluble antioxidants. Lower levels of dolichol are associated with enhanced peroxidation of lipids, which suggest that dolichol may have a protective role in the antioxidant machinery of cell membranes and perhaps be a key to understanding some adverse effects of statin therapy.

Changes in dolichol and pentosidine levels in the age-mismatched heterotopically transplanted rat heart

To address some basic questions about primary and secondary events in the process of aging in different cell and tissue types, we studied changes in the levels of biomarkers of the aging cells (dolichol) and connective tissue (pentosidine) in the heart of older (22-month-old) Lewis rats heterotopically transplanted in younger (3-month-old) syngenic recipients. Results showed that age-mismatched transplantation did not alter the age-related accumulation of dolichol and significantly reduced the accumulation of pentosidine in cardiac tissue. It is concluded that aging of heart muscle and connective tissues is controlled by two independent clocks; that accumulation of dolichol in older tissues may be a primary consequence of the process of aging, whereas the accumulation of pentosidine may be secondary, perhaps to changes in circulating cells endowed with advanced glycation end products-specific receptors; in the perspective of organ transplantation, the environment of a younger host may positively interact with the graft and rejuvenate its collagen.