Search PubMed⌕ Search

Biomedical subjects

A H Merrill

Publications and source records attributed to A H Merrill.

At least 19 recordsLinked to original sources
PubMed1995

Regulation of cytochrome P450 2C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis, and ceramides in rat hepatocytes.

Interleukin-1 triggers the down-regulation of several hepatic cytochrome P450 gene products, but the cellular signaling pathways involved are not known. We have examined the role of sphingomyelin hydrolysis to ceramide in the suppression of CYP2C11, a major constitutive form of cytochrome P450, by interleukin-1. Treatment of rat hepatocytes cultured on matrigel with interleukin-1 beta caused a rapid turnover of sphingomyelin and an increase in cellular ceramide, with no change in cellular phosphatidylcholine. The ceramide was composed mainly of a D-erythro-sphingosine backbone, suggesting that it was derived from sphingolipid hydrolysis rather than from increased de novo synthesis. Treatment of the cells with either N-acetyl-D-erythro-sphingosine (C2-ceramide) or bacterial sphingomyelinase suppressed the expression of CYP2C11 and induced the expression of the interleukin-1-responsive alpha 1-acid glycoprotein mRNA. In contrast, the acute-phase gene beta-fibrinogen, which is induced by interleukin-6 but not by interleukin-1, did not respond to C2-ceramide. N-Acetyl-D-erythro-sphinganine mimicked the effect of C2-ceramide on CYP2C11, but not on alpha 1-acid glycoprotein expression. These results are consistent with a role for ceramide or a related sphingolipid in mediating the down-regulation of CYP2C11, the induction of alpha 1-acid glycoprotein, and perhaps other cellular effects of interleukin-1 in hepatocytes.

Acute-Phase Proteins↗
PubMed1995

Differential roles of de novo sphingolipid biosynthesis and turnover in the "burst" of free sphingosine and sphinganine, and their 1-phosphates and N-acyl-derivatives, that occurs upon changing the medium of cells in culture.

Long-chain (sphingoid) bases are highly bioactive intermediates for sphingolipid metabolism, yet relatively little is known about how the amounts of these compounds are regulated. This study used J774A.1 cells to characterize the "burst" of sphinganine and sphingosine, or the transient increase of up to 10-fold in long-chain base mass, that occurs when cells in culture are changed to fresh medium. The increase in sphinganine was attributable to de novo sphingolipid biosynthesis because: 1) there is increased incorporation of [3H]serine and [3H]palmitate into sphinganine; 2) the incorporation of [3H]serine was equivalent to the increase in sphinganine mass; 3) beta-F-alanine, an inhibitor of serine palmitoyltransferase, blocked the sphinganine burst; 4) the magnitude of the burst depended on the concentration of serine in the medium, which is known to affect long-chain base biosynthesis; and 5) the appearance of sphinganine was relatively unaffected by lyso-osmotrophic agents (NH4Cl and chloroquine) that blocked sphingolipid hydrolysis in these cells. In contrast, the sphingosine burst arose mainly from turnover of complex sphingolipids because no incorporation of [3H]serine or [3H]palmitate into sphingosine was detected; sphingosine mass was not affected by beta-F-alanine or the serine concentration; and, the burst could be followed by the release of sphingosine and ceramide from complex sphingolipids (especially sphingomyelin) in a process that was inhibited by NH4Cl and chloroquine. Additionally, the fate of these long-chain bases differed: sphinganine was mostly (80-85%) acylated and incorporated into dihydroceramide and complex sphingolipids, whereas most of the sphingosine (70%) was phosphorylated and degraded, with incorporation of the resulting ethanolamine phosphate into phosphatidylethanolamine. Sphinganine, however, could be diverted toward degradation by adding an inhibitor of N-acylation (fumonisin B1). In accounting for the elevation in sphingosine and sphinganine after cells are changed to new medium, these studies have provided fundamental information about long-chain base metabolism. The existence of differential changes in sphinganine and sphingosine, as well as their 1-phosphates and N-acyl-derivatives, should be considered when evaluating the roles of sphingolipid metabolites in cell regulation.

Ammonium Chloride↗
PubMed1995

Sphingolipid biosynthesis de novo by rat hepatocytes in culture. Ceramide and sphingomyelin are associated with, but not required for, very low density lipoprotein secretion.

Sphingolipids are constituents of liver and lipoproteins, but relatively little is known about their synthesis and secretion. Incubation of rat hepatocytes with [14C]- or [3H]serine labeled the long-chain base backbones of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids were associated with the cells; however, 1-5% (the majority of which was ceramide) was released into the medium as part of very low density lipoproteins (VLDL). Since this is the first report that lipoproteins contain ceramide, lipoproteins were isolated from rat plasma, and the ceramide contents were (per mg of protein): 6.5 nmol for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins (1.018 < d < 1.063), 0.2 nmol for high density lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the albumin fraction; the lipoproteins also contained 0.1-0.4 nmol of free sphingosine/mg of protein. A number of factors affected the secretion of radiolabeled sphingolipids: 1) addition of palmitic acid, but not stearic or oleic acid, enhanced secretion due to an increase in long-chain base synthesis de novo. 2) Choline deficiency caused a 42% reduction in the secretion of radiolabeled sphingomyelin, but this was due to an effect on VLDL secretion rather than a decrease in sphingolipid synthesis. Removal of choline was examined because previous studies (Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004) have shown that choline deficiencies depress phosphatidylcholine synthesis and lipoprotein secretion. 3) Incubation of the cells with fumonisin B1, a mycotoxin inhibitor of sphinganine (sphingosine) N-acyltransferase, reduced overall sphingolipid synthesis and secretion by 90%, but had no effect on the secretion of apoB, phosphatidylcholine, or cholesterol. All together, these findings demonstrate that rat hepatocytes synthesize ceramide and sphingomyelin de novo and incorporate them into both cellular membranes and secreted VLDL, but normal sphingolipid synthesis is not required for lipoprotein secretion.

Animals↗
PubMed1995

Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1.

The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined. Fumonisin B1 inhibited the growth of yeast cells. Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner. The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells. Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1. Fumonisin B1 inhibited the synthesis of the inositol-containing sphingolipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids. The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined. Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined. However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase. These are key enzymes responsible for the synthesis of lipids in yeast. The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases.

Amidohydrolases↗
PubMed1995

Stereoselectivity of induction of the retinoblastoma gene product (pRb) dephosphorylation by D-erythro-sphingosine supports a role for pRb in growth suppression by sphingosine.

Sphingosine has been shown to inhibit cell growth in many cell lines although the mechanism of this effect remains obscure. More recently, D-erythro-sphingosine has been shown to act as an early inducer of dephosphorylation of the retinoblastoma gene product (pRb) in the lymphoblastic leukemia cell line MOLT-4 [Chao, R., Khan, W., & Hannun, Y.A. (1992) J. Biol. Chem., 267, 23459-23462]. In the current study, the role of the natural D-erythro-sphingosine in regulation of cell growth and pRb dephosphorylation was evaluated using chemically synthesized pure isomers of sphingosine. Of the four possible stereoisomers of sphingosine, D-erythro-sphingosine was most active in inducing dephosphorylation of pRb protein with an EC50% of 0.6 microM whereas its enantiomer L-erythro-sphingosine was 8-fold less potent with an EC50% of 5 microM. The dose responses for inhibition of cell growth were nearly identical to the EC50% for pRb dephosphorylation with D-erythro-sphingosine causing 50% inhibition at 0.6 microM whereas L-erythro-sphingosine was 5-6-fold less potent. All of the stereoisomers were taken up by the cells, and the greater potency of D-erythro-sphingosine was not due to differences in cellular uptake. The metabolism of D-erythro-sphingosine was also studied to evaluate the possible role of sphingosine metabolites on regulation of retinoblastoma protein. Evidence is provided against a role for ceramide or sphingosine 1-phosphate as mediators of the effects of sphingosine on pRb dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Death↗
PubMed1995

Fumonisin B1 alters sphingolipid metabolism and disrupts the barrier function of endothelial cells in culture.

Fumonisins are responsible for at least two diseases of veterinary importance (equine leukoencephalomalacia and porcine pulmonary edema) and are carcinogenic for experimental animals and, perhaps, humans. They have been found to disrupt sphingolipid metabolism in many types of cells, including hepatocytes, neurons, and renal cells. In this study, endothelial cells form porcine pulmonary arteries were cultured on micropore filters as a model for the endothelial barrier, and barrier function was quantitated as the movement of albumin across the endothelial monolayers. Fumonisin B1 increased the amount of free sphinganine by 20- to 30-fold within 3 hr, as expected for inhibition of sphinganine (sphingosine) N-acyltransferase by this mycotoxin. At 30 to 50 microM, fumonisin B1 doubled the rate of albumin transfer across endothelial monolayers; however, there was no loss of cell viability based on morphology or trypan blue exclusion. When 15 microM D-erythro-sphinganine was added to the cells, the rate of albumin transfer also doubled (after 24 hr incubation) without a loss of viability; however, this treatment increased the cellular level of sphinganine by > 100-fold. Addition of 25 microM sphinganine caused even greater albumin transfer, but also resulted in significant cell death. These results establish that fumonisin B1 and D-erythro-sphinganine allow accelerated passage of macromolecules across the endothelium. Fumonisin B1 alters sphingolipid biosynthesis with an elevation of sphinganine in the cells which may, at least in part, explain the observed disruption of endothelial barrier function.

Albumins↗
PubMed1995

Chronic toxicity of fumonisins from Fusarium moniliforme culture material (M-1325) to mink.

Adult female mink (Mustela vison) were fed a diet that contained Fusarium moniliforme culture material that provided dietary concentrations of 89 ppm fumonisin B1, 21 ppm fumonisin B2, and 8 ppm fumonisin B3 for 87 days. During the trial, there was mild lethargy in the mink fed fumonisins, but no other clinical signs or differences in feed consumption (measured during the first two weeks), body weights, or survivability were observed between the fumonisin-treated and control mink. Several hematologic parameters (mean corpuscular hemoglobin concentration, plasma total solids, and lymphocyte concentration) and serum chemical concentrations (globulin, phosphorus, potassium, blood urea nitrogen, creatinine, bilirubin, and cholesterol) and activities (alkaline phosphatase, alanine aminotransferase, amylase, and aspartate aminotransferase) were greater in the mink fed fumonisins than in the controls. Serum albumin/globulin and sodium/potassium ratios and chloride concentrations were lower in the fumonisin-fed mink than in the controls. The concentrations of free sphinganine and the ratio of free sphinganine to free sphingosine in the liver and kidneys of the fumonisin-treated mink were greater than in the control mink. No histopathologic alterations were associated with fumonisin treatment. These results indicate that long-term dietary exposure to F. moniliforme culture material containing 118 ppm total fumonisins is not lethal to adult mink, but can produce adverse physiological effects in the animals.

Animals↗
PubMed1995

Fumonisin B1 alters sphingolipid metabolism and immune function in BALB/c mice: immunological responses to fumonisin B1.

Fumonisins have been reported to have diverse effects on animals, including immunosuppression in chickens and feeder calves; therefore, the effects of fumonisin B1 (FB1) on immune function in BALB/c mice was investigated. When administered i.p. with sheep red blood cells (SRBC), 5 to 100 micrograms of FB1 reduced the number of plaque-forming cells (PFC) produced against SRBC; however, when administered daily, 1 to 50 micrograms of FB1 caused a 4 to 12-fold increase in the number of PFC after SRBC injection. Therefore, FB1 is not only immunosuppressive; but also, immunostimulatory. To test the possibility that there may have been an immune response to FB1 as an antigen, FB1 was injected into mice and the number of splenic cells forming rosettes on FB1-treated SRBC was determined. There were dose-dependent increases in the antigen-binding cells, with up to 4.9- and 4.6-fold increases, respectively, upon primary and secondary immunization. FB1-binding immunoglobulins could be detected in sera from some treated mice, but this response was not obtained in every experiment. In summary, these results show that FB1 has diverse effects on the immune system, causing both stimulation and suppression of the response to foreign antigens, and apparently inducing an antigenic response to FB1.

Adjuvants, Immunologic↗
PubMed1995

Role of dietary sphingolipids and inhibitors of sphingolipid metabolism in cancer and other diseases.

Sphingolipids are found in all eukaryotic and some prokaryotic organisms and participate in the regulation of cell growth, differentiation, and diverse cell functions including cell-cell communication, cell-substratum interactions and intracellular signal transduction. Nonetheless, the field of nutrition has given scant attention to these compounds so that little is known about the following fundamental questions: What is the fate of sphingolipids that are consumed in food? Does consumption of dietary sphingolipids affect the behavior of cells in the gastrointestinal tract or other organs? How do other factors in the diet affect sphingolipid metabolism? Several recent findings underscore the importance of these questions, for examples: 1) Sphingolipids are digested throughout the GI tract to ceramide and sphingosine, which are highly bioactive compounds that affect cellular regulatory pathways; 2) addition of sphingomyelin to a standard AIN diet (which is essentially devoid of sphingolipids) reduces the appearance of aberrant colonic crypts, and perhaps the number of tumors, in mice treated with a colon carcinogen; and 3) an enzyme of sphingolipid metabolism has been discovered to be the target of a class of toxic and carcinogenic mycotoxins called fumonisins. Given these recent findings, it is possible that some of the confusion that has arisen regarding the relationships between dietary fat and disease might be due to the lack of consideration of the sphingolipids that are also present.

Animals↗
PubMed1992

Influences of sphingosine on two-stage skin tumorigenesis in Sencar mice.

Sphingosine (SPH) was studied as an inhibitor of skin tumor promotion in skin cancer initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Two tumorigenesis studies were conducted using female Sencar mice treated with 10 nmol DMBA in 0.2 ml acetone at 8 weeks of age and promoted beginning 1 week later with 3.2 nmol TPA applied twice per week. In the high-dose study, 10 mumol SPH was applied 30 min before each TPA treatment. The low-dose study used 0.5, 0.05, or 0.01 mumol treatments with SPH 30 min before TPA. In the high-dose study SPH treatment alone following initiation by DMBA, and SPH treatment preceding TPA in DMBA-initiated mice accelerated the development of papillomas in comparison with the DMBA/TPA-treated group. The low-dose experiment showed no consistent alteration of tumorigenesis by SPH in the DMBA/TPA-treated groups, and low doses of SPH following DMBA did not promote skin tumorigenesis. SPH treatment did not alter body weight in either experiment.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

In vitro toxicology of fumonisins and the mechanistic implications.

The effects of fumonisins B1(FB1), B2(FB2), and the backbone of fumonisin B1 remaining after hydrolysis of the tricarballylic groups with base (HFB1) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK1). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC50 of FB1 = 0.1 microM), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC50 for FB1 = 35 microM), and caused decreased cell proliferation as well. Higher doses (greater than or equal to 70 microM) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.

Animals↗
PubMed1992

Fumonisin inhibition of de novo sphingolipid biosynthesis and cytotoxicity are correlated in LLC-PK1 cells.

Fumonisins are a group of structurally related compounds produced by Fusarium moniliforme. Recently, it has been shown that fumonisins B1 and B2 are the first naturally occurring inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase) in rat primary hepatocytes (Wang et al. J. Biol. Chem. 266, 14, 486-14, 490, 1991). These enzymes are key components in the pathways for de novo sphingolipid biosynthesis and sphingolipid turnover. The results of the present study show that fumonisins B1 and B2 inhibit proliferation and are cytotoxic to LLC-PK1 cells. Concentrations of fumonisin B1 and B2 between 10 and 35 microM inhibited cell proliferation, whereas higher concentrations (greater than 35 microM) killed cells. Inhibition of cell proliferation and cell death were preceded by a lag period of at least 24 hr during which cells appeared to be functioning normally. Cells exposed to fumonisin B1 exhibited normal growth kinetics and morphology soon after fumonisin B1 was removed; thus, the effects of fumonisin B1 were reversible. The EC50 for alterations in sphingolipid biosynthesis was 10 to 15 microM. Inhibition of de novo sphingolipid biosynthesis occurred before inhibition of cell proliferation or cytotoxicity, and the dose response for the decrease in the [3H]sphingosine to [3H]sphinganine ratio at 7 hr closely paralleled the dose response for effects on proliferation and cytotoxicity at 3-5 days. In addition, the level of free sphinganine, and to a lesser extent sphingosine, increased in fumonisin-treated cells in a dose-dependent manner. During the 24-hr lag period preceding inhibition of cell proliferation, the free sphinganine content increased by 12,800% in cells exposed to 35 microM fumonisin B1. Whereas a mechanistic relationship between the inhibition of de novo sphingolipid biosynthesis and inhibition of proliferation and cell death has not been demonstrated, the results of this study support the hypothesis that inhibition of de novo sphingolipid biosynthesis is an early event in the toxicity of fumonisins to LLC-PK1 cells.

Acyltransferases↗
PubMed1992

Increases in serum sphingosine and sphinganine and decreases in complex sphingolipids in ponies given feed containing fumonisins, mycotoxins produced by Fusarium moniliforme.

Consumption of food contaminated with Fusarium moniliforme causes leucoencephalomalacia and hepatotoxicity in horses, pulmonary edema in pigs and liver cancer in rats, and has been correlated with esophageal cancer in humans. The causative agents are thought to be a family of compounds called fumonisins, which have recently been shown to be potent inhibitors of sphingosine (sphinganine) N-acyltransferase. Because inhibition at this step blocks the formation of complex sphingolipids while leading to accumulation of sphinganine, we hypothesized that exposure of animals to fumonisin-contaminated feed might be detected by analyses of serum sphingolipids. Within days of giving ponies feed contaminated with 15 to 44 micrograms/g fumonisin B1, there was an increase in the amount of free sphinganine (and sometimes sphingosine) and a reduction in complex sphingolipids. Free sphinganine and sphingosine decreased when ponies consumed less of the contaminated feed, and increased again when they consumed more fumonisin. When toxicosis was evident as indicated by other serum markers, complex sphingolipids as well as free sphingosine and sphinganine were elevated, probably due to loss of sphingolipids from dying cells. These findings establish that consumption of fumonisin-contaminated feed disrupts sphingolipid metabolism. Because the changes in sphinganine and sphingosine were seen before liver enzymes were noticeably elevated, they may be an early marker of exposure to fumonisins.

Animal Feed↗
PubMed1992

Ceramide: a new lipid "second messenger"?

1-Alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, and gamma-interferon induce sphingomyelin turnover to ceramide in HL-60 cells. The time frame of the turnover is consistent with ceramide formation being a mediator of growth and differentiation; furthermore, differentiation can be induced by addition of exogenous ceramides alone. Hence, the action of the biologically active form of vitamin D and other nutritionally relevant factors may be mediated by lipid second messenger pathways involving sphingolipids.

Animals↗
PubMed1992

Analyses of plasma for metabolic and hormonal changes in rats flown aboard COSMOS 2044.

Plasma samples from rats flown aboard COS-MOS 2044 were analyzed for the levels of key metabolites, electrolytes, enzymes, and hormones. The major differences between the flight group and the synchronous control were elevations in glucose, cholesterol, phosphate, creatinine, blood urea nitrogen, lactate dehydrogenase, and aspartate amino-transferase and decreased levels of thyroxine. Most of these differences were not mimicked by tail suspension of ground-based rats; however, both flight and suspended rats exhibited inhibited testosterone secretion. Corticosterone, immunoreactive growth hormone, and prolactin showed inconsistent differences from the various control groups, suggesting that the levels of these hormones were not due to actual or simulated microgravity.

Adrenal Glands↗
PubMed1992

Differences in glycogen, lipids, and enzymes in livers from rats flown on COSMOS 2044.

Livers from rats flown aboard COSMOS 2044 were analyzed for protein, carbohydrate (glycogen), and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. The major differences between the flight group and the synchronous control were elevations in microsomal protein, liver glycogen content, tyrosine aminotransferase, and tryptophan oxygenase and reductions in sphingolipids and the rate-limiting enzyme of heme biosynthesis, delta-aminolevulinic acid synthase. These results provide further evidence that spaceflight has pronounced and diverse effects on liver function; however, some of the results with samples from COSMOS 2044 differed notably from those from previous spaceflights. This may be due to conditions of spaceflight and/or the postflight recovery period for COSMOS 2044.

Amino Acids↗
PubMed1991

Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme.

Culture materials and grains contaminated with certain isolates of Fusarium moniliforme cause equine leucoencephalomalacia, porcine pulmonary edema syndrome, and liver cancer in rats. The causative agents are thought to be a family of compounds called fumonisins, which bear considerable structural similarity to the long-chain (sphingoid) base backbones of sphingolipids. Incubation of rat hepatocytes with fumonisins inhibited incorporation of [14C]serine into the sphingosine moiety of cellular sphingolipids with an IC50 of 0.1 microM for fumonisin B1. In contrast, fumonisin B1 increased the amount of the biosynthetic intermediate sphinganine, which suggests that fumonisins inhibit the conversion of [14C]sphinganine to N-acyl-[14C]sphinganines, a step that is thought to precede introduction of the 4,5-trans double bond of sphingosine (Merrill, A.H., Jr. and Wang, E. (1986) J. Biol. Chem. 261, 3764-3769). In agreement with this mechanism, fumonisin B1 inhibited the activity of sphingosine N-acyltransferase (ceramide synthase) in rat liver microsomes with 50% inhibition at approximately 0.1 microM and reduced the conversion of [3H]sphingosine to [3H]ceramide by intact hepatocytes. As far as we are aware, this is the first discovery of a naturally occurring inhibitor of this step of sphingolipid metabolism. These findings suggest that disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases that have been associated with consumption of fumonisins.

Acyltransferases↗