Age-related changes of isoprenoid biosynthesis in rat liver and brain.

The physiological role of dolichol is not yet known but its accumulation in several tissues has been extensively reported in various physiological states or pathological conditions. Increased dolichol concentration in mammalian tissues during ageing has been also reported; in particular, we have previously indicated dolichol accumulation in liver as a new biomarker of ageing. However, the mechanism and the role of this accumulation is unknown. The aim of this work was to study the mechanism of the age-dependent dolichol accumulation analysing, in the liver and in the brain, the activity of the rate-limiting enzyme of isoprenoid biosynthesis, the 3-hydroxy 3-methylglutaryl CoA reductase, the dolichol and cholesterol synthesis on aged rats both fed ad libitum and caloric restricted. Furthermore, the dolichol and cholesterol levels in the plasma were assayed. The data shows that during ageing, the tissue dolichol accumulation is connected with the increase of 3-hydroxy 3-methylglutaryl CoA reductase activity and only in liver affected by diet restriction. In addition the aged rats maintain the capability to regulate their tissue cholesterol content by modifying cholesterol delivery into the blood. The amount of the 3-hydroxy 3-methylglutaryl CoA reductase enzyme detectable in liver and brain by Western blot analysis does not show significant changes during ageing. The presented data show that the accumulation of dolichol is related to the loss of enzymatic regulation characteristic of ageing. In fact, a higher mevalonate availability deriving from an increased expressed activity of HMGCoA-R could cause an increased production of dolichol.

Estradiol-induced IP(3) mediates the estrogen receptor activity expressed in human cells.

The recent findings that estradiol-induced IP(3)/PKC-alpha signalling pathway triggers DNA synthesis in HepG2 cells, containing estrogen receptor unable to stimulate gene transactivation, raises the hypothesis that this pathway represents an alternative signalling present when the amount of estrogen receptor (ER) is insufficient to mediate genomic effects. beta-estradiol-stimulated DNA synthesis and target gene expression have been studied in HepG2 and, ER-alpha or ER-beta negative, HeLa cells. We also examined whether either receptor is required for rapid effects of estrogen on DNA synthesis. Finally, the consequences of increased ER expression on estrogen-induced DNA synthesis and synthetic target gene expression have been evaluated. Our data indicate that the E2-induced IP(3) production is dependent on expression of either ER-alpha or ER-beta in both HepG2 and HeLa cells. Moreover, inhibition of the IP(3) second messenger pathway blocks E2-induced cellular actions suggesting that this second messenger is responsible for estrogen's rapid, non-genomic effects on both DNA synthesis and gene expression.

Nitric oxide inhibits selectively the 17beta-estradiol-induced gene expression without affecting nongenomic events in HeLa cells.

17beta-Estradiol (E2) induces genomic (i.e., pC3-luciferase promoter-reporter construct expression) and nongenomic (i.e., DNA synthesis and IP(3) production) effects in HeLa cells only after transient transfection with the human estrogen receptor alpha (ERalpha) reporter plasmid. Here the effect of nitric oxide (NO) on both E2-induced effects in transiently transfected HeLa cells is reported. Remarkably, the E2-dependent gene transcription is inhibited dose-dependently by NO. By contrast, DNA synthesis and IP(3) production, representing nongenomic E2-dependent effects, are unaffected by NO. The selective NO action on E2-induced functions may be related to NO-mediated chemical modification(s) of the Cys residues present in the DNA recognition domain of ERalpha impairing DNA binding.

Activation of IP(3)-protein kinase C-alpha signal transduction pathway precedes the changes of plasma cholesterol, hepatic lipid metabolism and induction of low-density lipoprotein receptor expression in 17-beta-oestradiol-treated rats.

The intracellular concentration of cholesterol is regulated by the balance between endogenous synthesis and exogenous uptake. Oestrogens have been reported to be involved in the physiological regulation of cellular cholesterol content. Relevant reports have focused on long-term responses and there is a lack of information about the relationship between the timing of the oestrogen effect and the regulation of cholesterol homeostasis. The aim of this work has been to set up a systematic picture of the short-term effects induced by oestrogen on hepatic lipid metabolism in vivo and the involvement of some relevant signal transduction pathways. At intervals after oestrogen administration (30 min to 6 h), oestrogen receptor expression and changes in liver cAMP, IP(3) and protein kinase C-alpha (PKC-alpha) were followed. Changes in the expression of the low density lipoprotein receptor at mRNA and protein levels, and of hydroxy-methyl-glutaryl-CoA reductase activity have been verified. At the same time, the content of hepatic cholesterol, ubiquinone and dolichol and of plasma cholesterol have been determined. Changes of rab 5 and rab 8, small GTP-binding prenylated proteins involved in the transfer of neosynthesised proteins through the cell, have been also checked. In vivo treatment with oestradiol produced no change in cyclic AMP but a rapid increase in IP(3), increased PKC-alpha localisation on the membranes and enhanced expression of the low density lipoprotein receptor in the liver occurred. PKC inhibition completely prevented any increase in low density lipoprotein receptor mRNA in isolated and perfused rat liver. Early changes of ubiquinone and dolichol content and a later reduction in hepatic hydroxy-methyl-glutaryl-CoA reductase activity and plasma cholesterol content were also detectable. A functional role of the IP(3) -protein kinase C-alpha pathway in the induction of the low density lipoprotein receptor is suggested. Experimental Physiology (2001) 86.1, 39-45.

beta-estradiol stimulation of DNA synthesis requires different PKC isoforms in HepG2 and MCF7 cells.

The role exerted by protein kinase C (PKC) on estrogen-induced DNA synthesis has been investigated in hepatic and mammary gland cells, HepG2 and MCF7. 17-beta-estradiol stimulated DNA synthesis in HepG2 and MCF7 cells, maximal effect occurring at 10 nM. DNA synthesis stimulation was prevented by anti-estrogen ICI 182,780 and by inhibitor of PKC, Ro 31-8220. The rapid estradiol effects in MCF7 cells were determined by following the inositol trisphosphate (IP(3)) production and PKC-alpha membrane translocation. After estradiol treatment the increase of IP(3) production, prevented by anti-estrogen or by phospholipase C (PLC) inhibitor (neomycin), was present in MCF7 cells. In MDA cells, devoid of estrogen receptor, no effect was observed. The PKC-alpha presence on the membranes appeared unchanged in MCF7 cells. The PLC inhibitors, neomycin and U73,122, and PKC-alpha down regulator, phorbol 12-myristate 13-acetate (PMA), were able to prevent estradiol-induced DNA synthesis in hepatoma cells, but ineffective in mammary cells; wortmannin, an inhibitor of phosphoinositide 3-kinases (PI3-K), blocked DNA synthesis in both cell lines. These data show that beta-estradiol, via an estrogen receptor-mediated mechanism, activates more signal transduction pathways, and consequently different PKC isoforms in two responsive cell lines. In both cell lines PI3-K/PKC pathway is functional to the estrogen regulation of DNA synthesis, whereas in HepG2 cells the parallel involvement of the PLC/PKC-alpha pathway is present. The reported results indicate that the DNA synthesis stimulation by beta-estradiol requires the estrogen receptor and utilises one or more activated pathways in dependence on the cell equipment.

Enhanced prenyltransferase activity and Rab content in rat liver regeneration.

Rabs are small GTP-binding proteins with a regulatory role in intracellular vesicular traffic. The modulation of their levels and activity in different physiological situations is poorly understood. During the first cell cycle of rat liver regeneration we observed a differential regulation of some Rabs, with a progressive increase of those involved in exocytosis and a progressive decrease of one involved in endocytosis. This could be related with the need of exposing growth factor receptors and prolonging the transduction of their signal in preparation for mitosis. Moreover, we observed an increased activity of protein prenyltransferases, the enzymes responsible for the prenylation of several proteins involved in crucial processes of proliferation, without a corresponding increase in the amount of prenyltransferase protein.

The promoter of the rat 3-hydroxy-3-methylglutaryl coenzyme A reductase gene contains a tissue-specific estrogen-responsive region.

The isoprenoid metabolic pathway is mainly regulated at the level of conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA reductase. As estrogens are known to influence cholesterol metabolism, we have explored the potential regulation of the HMG CoA reductase gene promoter by estrogens. The promoter contains an estrogen-responsive element-like sequence at position -93 (termed Red-ERE), which differs from the ERE consensus by one mismatch in each half of the palindrome. A Red-ERE oligonucleotide specifically bound estrogen receptor in vitro and conferred receptor-dependent estrogen responsiveness to a heterologous promoter in all cell lines tested. However, expression of a reporter driven by the rat HMG CoA reductase promoter was induced by estrogen treatment after transient transfection into the breast cancer cell line MCF-7 cells but not in hepatic cell lines expressing estrogen receptor. Estrogen induction in MCF-7 cells was dependent on the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384. A functional cAMP-responsive element is located immediately upstream of the Red-ERE, but cAMP and estrogens inhibit each other in terms of transactivation of the promoter. Similarly, induction by estrogens was inhibited by micromolar concentrations of cholesterol, likely acting via changes in occupancy of the sterol-responsive element located 70 bp upstream of the Red-ERE. Thus, within its natural context, Red-ERE is able to mediate hormonal regulation of the HMG CoA reductase gene in tissues that respond to estrogens with enhanced cell proliferation, while it is not operative in liver cells. We postulate that this tissue-specific regulation of HMG CoA reductase by estrogens could partially explain the protective effect of estrogens against heart disease.

Estrogen stimulates intracellular traffic in the liver of Rana esculenta complex by modifying Rab protein content.

During vitellogenesis in oviparous animals, estrogens induce the synthesis of the yolk precursor vitellogenin, a lipophosphoprotein rich in cholesterol. Estrogens also induce the activity of 3-hydroxy-3-methylglutaryl CoA reductase, that is necessary for the lipidation of vitellogenin. This increased enzyme activity could also be important for the production of isoprenoid groups that post-translationally modify proteins such as the Rab proteins, which are small G proteins involved in intracellular traffic. The effect of estrogens on the production of prenylated proteins and on the levels of Rab proteins in the liver of Rana esculenta complex has been studied. An increase of the Rabs specifically involved in the exocytic pathway was observed and is probably related to the need for export of massive amounts of newly synthesized vitellogenin.

Estrogens cause rapid activation of IP3-PKC-alpha signal transduction pathway in HEPG2 cells.

The mechanisms through which steroids affect target cells are not fully understood. In addition to the classic model, there is now increasing evidence that steroids can exert rapid actions. It must still be elucidated if rapid and slow estrogen actions produce co-operative and/or integrative functions. The effects of estrogen on inositol trisphosphate (IP3) production and PKC-alpha levels on membrane in the HEPG2 cell line have been investigated. Results show that estrogen addition to HEPG2 cells causes a rapid increase of IP3 production. The effect was totally inhibited by pre-incubation with tyrosine-kinase inhibitor genisteine and with the anti-estrogen ICI 182,780. An increased PKC-alpha level on the membrane fraction was present 30 min after estrogen exposure. The strong signal could elicit a variety of cellular responses such as modulation of ion channel, stimulation of cell proliferation, and phosphorylation of cytosolic ER. The ability of estrogen to trigger IP3 production in human hepatoma cells is a novel aspect of estrogen action that requires the current model of hormone stimulation target cells to be revised.

Age-dependent accumulation of dolichol in rat liver: is tissue dolichol a biomarker of aging?

Dolichols are long hydrophobic molecules broadly distributed in all tissues and cellular membranes of eukariotic cells. Dolichol affects membrane structure and fluidity, membrane-associated protein activities, and membrane sensitivity to oxidative stress. Reports have shown that dolichols exhibit a remarkable (6- to 30-fold) age-related increase in the tissues of adult and mature rats and of old flies, mice, and humans. In our longitudinal study, the age-related accumulation of dolichol was monitored in the liver tissue of male Sprague Dawley rats fed ad libitum up to age of 27 months. In addition 24-month-old rats subjected to different regimens of anti-aging diet restriction (40% calorie restriction or every-other-day feeding ad libitum) were tested. A parallel study of the accumulation of carbonyl in liver protein (a proposed biomarker of aging) was made. In addition, the age-related decline of liver autophagy/proteolysis was studied in isolated liver cells, in view of the essential role of this function in liver membrane maintenance. Results show that an age-dependent accumulation of dolichol can be observed in the liver of the rats fed ad libitum but not in the liver of 24-month-old food-restricted rats, that accumulation of dolichol precedes the accumulation of altered liver proteins, and that dolichol accumulation is accompanied by a decline in liver autophagy. It is concluded that dolichol accumulation satisfies the proposed primary and secondary applicable criteria and the desirable features required to be qualified as a biomarker of aging.

Nuclear lamina assembly in the first cell cycle of rat liver regeneration.

The nuclear lamina is a fibrous structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of its protein components, the lamins A, B, and C. The isoprenylation of the lamins is essential for their proper membrane anchoring and functionality. The content and the membrane association of nuclear lamins and the subcellular localization at light and electron microscopical levels were studied at different times of rat liver regeneration. This model for the good synchrony of the first cell cycle is particularly suited for the study of cell-cycle-dependent modifications and is particularly interesting for the increased protein prenylation found in S phase. The biochemical results show an increased lamin content in nuclear proteins in G1 phase and a decreased content in M phase, along with an enhanced cytosolic localization of A and C lamins at later stages. The morphological results show in M phase, also in nondividing cells, a decreased lamin-like immunoreactivity around the nucleus with an apparent nuclear lamina disassembly. These data emphasize the dynamic organization of nuclear lamina not only in mitosis but also in interphase. The reduction and partial solubilization of nuclear lamina in M phase suggest a reorganization of the nuclear envelope also in those cells that do not appear in mitosis but have replicated their DNA content that will result in a higher degree of polyploidy.

Independent responsiveness of frog liver low-density lipoprotein receptor and HMGCoA reductase to estrogen treatment.

Cholesterol metabolism in the female frog exhibits circannual modifications which parallel plasma estrogen fluctuations. Estrogens enhance production and lipidation of the yolk precursor vitellogenin by inducing the transcription of its gene and by stimulating the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. The time dependence of the effects that these hormones have on HMGCoA reductase and the low-density lipoprotein (LDL) receptor in the liver of Rana esculenta complex has been investigated. Following estrogen treatment, the levels of LDL receptor mRNA and protein gradually increased, with a maximum concentration observed at 3 days. The HMGCoA reductase protein level increased progressively, while the mRNA level was not significantly modified. Thus the LDL receptor and HMGCoA reductase in frog behave independently after estrogen stimulation, as already reported to occur in the rat. This suggests a uncoordinated regulation that might be even partially related with the changes in cellular cholesterol content.

Independent behavior of rat liver LDL receptor and HMGCoA reductase under estrogen treatment.

The main molecules of hepatic cholesterol homeostasis are HMGCoA reductase, the key enzyme of the biosynthetic pathway, and LDL receptor, responsible for the uptake of plasma lipoproteins. Estrogens are reported to cause hypolipidemia in mammalians inducing hepatic LDL receptor. The effect of such hormones on HMGCoA reductase is very ambiguous. The mechanism and the time-dependence of the effects of these hormones on HMGCoA reductase and LDL receptor in rat liver have been investigated at mRNA and protein levels, at different times after estrogen administration. Estrogens cause an early increase of LDLr, at both mRNA and protein level, and an increase of HMGCoA reductase, just at protein level, detectable only after 5 days. The independent behavior of LDLr and HMGCoA reductase under estrogen treatment suggests a not coordinate regulation by these hormones.

Win/win with an improved appeals process.

In the past, decisions on what services were appropriate and/or desirable were made between the patient and the physician. In most cases, the cost of services was ignored. Lately, concern for cost containment has introduced a new person into the health care decision-making process: the managed care monitor/planner. The appearance of this new person has produced ambivalent feelings among patients and physicians, from joyful approval for those concerned with rising costs to extreme anger for those whose services are denied, while perceived by them as absolutely necessary. Thus, appeal mechanisms have become a way of life. This article explores ways in which the appeals process may be used as a tool to improve satisfaction levels among providers and subscribers and still fulfill the cost containment and efficiency goals of case management.

Utilization management, case management, and you.

Historically, most monitoring functions have been carried out by insurance companies. Monitoring costs was considered their fiduciary obligation to their customers. The exercise of this fiduciary obligation kept premiums low, while increasing or maintaining the benefit levels. Risk (the assumption of losses generated by services costing more than the income received from premiums) was assumed by the insurance company and eventually passed to the customer or the payer. Today, risk is being transferred more and more to the provider. This transfer was started by the creation of DRGs, the main purpose of which was to transfer risk from payers (insurance companies, employers, state and federal government, etc.) to provider health care organizations (physician groups, individual practitioners, hospitals, clinics, etc.).

Economic credentialing is here to stay.

Health care organizations are being scrutinized by payers for the efficiency of their processes to render health care. Organizations must offer alternative avenues to satisfy health care needs that are less resource consuming and have a reasonable chance of success. This presents an enormous challenge to U.S. health care. In the past, while in training, physicians were conditioned to ignore costs in the provision of care. We cannot afford that behavior today. Physicians must be reeducated and their behavior reconditioned to alter the teachings at medical school and residency with respect to resource utilization. To be effective, this education and behavior modification must be done in a nonpunitive fashion.

Constitutional rights versus malpractice insurance settlements.

The increasing costs and complexity of malpractice litigation have created an statutory right that allows malpractice insurance companies to settle malpractice claims regardless of the desires of the defendant physician. In the past, the consequences of settling a malpractice claim out of court were not as important as they are today. The Health Care Quality Improvement Act of 1986 mandates that any settlement in behalf of a physician be documented in the National Practitioner Data Bank (NPDB), which must be consulted every time the physician is credentialed. This NPDB requirement denies due process to health care providers and thus becomes a violation of the federal and many state constitutions. Physician executives and medical leaders must bring these issues to the table and negotiate solutions before damage to practicing physicians and the U.S. health care delivery system caused by this legal paradox become too severe.

Changes in plasma dolichol levels, transport, and hepatic delivery during rat liver regeneration.

During the proliferative process that follows partial hepatectomy in the rat, the dolichol content increases in both plasma and liver. Its transport in the blood by lipoproteins also changes. The difference in the distribution of dolichols of various chain lengths in plasma and in the liver is further enhanced during liver regeneration. The dolichol released by perfused liver shows a homologue distribution more similar to that observable in blood than in the liver, thus confirming the importance of the liver as a regulatory site for the blood dolichol supply.