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Hypocholesterolemic properties of nitric oxide. In vivo and in vitro studies using nitric oxide donors
Biochimica et Biophysica Acta 1392 Ž1998. 41–50
Hypocholesterolemic properties of nitric oxide. In vivo and in vitro studies using nitric oxide donors E.M. Kurowska ) , K.K. Carroll Department of Biochemistry, Centre for Human Nutrition, UniÕersity of Western Ontario, London, Ontario, Canada, N6A 5C1 Received 18 August 1997; revised 17 December 1997; accepted 19 December 1997
Abstract Previous results suggested that changes in the activity of nitric oxide ŽNO. can influence metabolism of apo B-containing lipoproteins. Therefore, we studied effects of exogenous NO donors and physiological NO precursors on metabolism of these lipoproteins. In rabbits, addition of 0.03% sodium nitroprusside ŽNaNP. to a semipurified, cholesterol-free, casein diet counteracted the elevation of LDL cholesterol induced by this diet but did not alter liver lipids after 4 weeks of feeding. In HepG2 cells, addition of nontoxic concentrations of another NO donor, S-nitroso-N-acetylpenicillamine ŽSNAP. to culture medium caused a dose-dependent reduction of medium apo B after 24 h. At the concentration 0.5 mM, SNAP significantly decreased medium apo B by 50% without altering total synthesis and secretion of proteins and without altering rates of cellular sterol synthesis. In cells incubated with L-arginine, reduction of medium apo B was not associated with increased NO production whereas in those exposed to N–OH–Arg medium apo B levels were not altered. We concluded that synthetic NO donors can reduce hypercholesterolemia by affecting apo B metabolism directly in the liver, via the sterol-independent mechanism. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide donor; Cholesterol; HepG2 cell; ŽRabbit.
1. Introduction There is a growing body of evidence that nitric oxide ŽNO., the endothelium-derived relaxing factor originating from metabolism of L-arginine, can prevent atherosclerosis by inhibiting oxidation of lipoproteins in the arterial wall w1x. In addition, some recent studies have suggested that NO can modulate metabolism of lipoproteins when its availability is altered by administration of synthetic NO synthase inhibitors or NO donors. In cholesterol-fed rabbits, )
Corresponding author. Fax: q1-519-661-4006; E-mail: [email protected]
chronic administration of the NO synthase inhibitor, N v-nitro-L-arginine ŽL-NAME., promoted atherosclerosis and also tended to increase hypercholesterolemia w2x. Similarly, serum cholesterol level was moderately elevated in rats treated with another NO synthase inhibitor, L-N v Nitroarginine ŽL-NNA. w3x. Conversely, a substantial, dose-dependent reduction of hypercholesterolemia as well as atherosclerosis was observed in cholesterol-fed Japanese quail after chronic oral administration of an NO donor, sodium nitroprusside ŽNaNP. w4x. A relationship between activity of NO and cholesterolemia has also been found in the absence of pharmacological intervention. In rabbits with diet-in-
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 2 1 5 - 4
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E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
duced hypercholesterolemia, urinary excretion of NO metabolic products, nitrites, was decreased w5x and plasma levels of an endogenous NO synthase inhibitor, N G, N G-dimethylarginine, were increased, suggesting impaired NO activity w6,7x. In agreement, an excessive release of NO during inflammation and tissue injury has been associated with acquired hypocholesterolemia, although this response has been postulated to be at least partly due to preceding release of cytokines w8x. Previous results suggested that NO-mediated hypocholesterolemic responses are unlikely to be enhanced by increased dietary intake of NO precursor, L-arginine. Dietary supplementation with L-arginine has been shown to reverse endothelial dysfunction caused by hypercholesterolemia and to inhibit atherogenesis w9x but failed to counteract hypercholesterolemia itself w5,10,11x, except when the endogenous pool of L-arginine was severely depleted by prior chronic administration of NO inhibitors w3x. In contrast, in our recent studies, dietary L-arginine appeared to have anti-hypercholesterolemic properties in rabbits fed hypercholesterolemic amino acid diets as well as apo B-lowering properties in HepG2 cells w12,13x. However, in the rabbit model, feeding high levels of L-arginine was not associated with increased plasma and liver content of NO end metabolites, nitrates Ž unpublished data. . Another possibility, that a stable intermediate precursor in L-arginine-NO pathway, N G-hydroxy-L-arginine ŽN–OH–Arg. , could be important in NO-mediated regulation of lipoprotein metabolism, is yet to be investigated. It has been shown that in vitro, N–OH–Arg can be oxidized to NO and nitrite in the absence of active NO synthase, probably by cytochrome P450 w14x. The mechanism by which changes in activity of NO can influence the metabolism of lipoproteins is poorly understood. The lipoprotein responses produced in animals treated with inhibitors of NO synthase, although obscured by cholesterol feeding, suggested that VLDL andror LDL rather than HDL cholesterol are likely to be affected w2x. Since VLDL and LDL are synthesized and catabolized in the liver, we hypothesized that regulation of lipoprotein metabolism by NO, directly or via its intermediate precursors, L-arginine or N–OH–Arg, could occur in this organ. To understand the nature of this regulation better, we investigated effects of exogenous NO
donors on metabolism of lipoproteins in vivo and in vitro. In vivo, effects of 0.03% NaNP supplementation on lipoprotein profile and on liver lipids was tested using rabbits in which experimental hypercholesterolemia associated with elevation of LDL cholesterol, similar to that in humans, was induced by feeding a semipurified, cholesterol-free, casein diet w15x. In vitro, cholesterolemic responses were analyzed in human hepatoma ŽHepG2. cells after their exposure to a less toxic NO donor, S-nitroso-Nacetylpenicillamine ŽSNAP.. Experiments were also conducted to determine whether in HepG2 cells, the apo B-lowering effect induced by incubation with high levels of L-arginine could be mediated by NO and whether a similar reduction of apo B in the medium could be obtained by exposure of HepG2 cells to N-OH-Arg.
2. Materials and methods 2.1. Animal experiment The animal protocol was in accordance with the Canadian Council of Animal Care guidelines and was approved by the Council of Animal Care, University of Western Ontario. Young New Zealand White male rabbits ŽReimen’s Fur Ranches, Guelph, Ontario, Canada., weighing 1.6–1.7 kg, were used in these experiments. The animals were housed individually in galvanized cages with wire bottoms, in a room maintained at 21–248C with a 12-h light:dark cycle. Following adaptation period w15x, they were randomly divided into two groups, 6 animals each, and given low-fat, cholesterol-free, semipurified diet containing 25% casein, with or without addition of 0.03% NaNP, for a period of 4 weeks. The composition of the casein diet was described previously w15x. Food and water were provided ad libitum. Weight gains and food consumption were monitored weekly. At the end of the study, food was withdrawn, the animals were killed 16–18 h later by an overdose of Euthanyl Forte ŽCanada Packers, Cambridge, Ontario, Canada. and blood samples were taken by heart puncture. Total and free cholesterol in serum and lipoprotein fractions were measured with enzymatic kits ŽCHOD-PAP and F-CHOL, Boehringer-Man-
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
nheim, Montreal, Canada.. VLDL Ž d - 1.006 grml., LDL Ž 1.006 - d - 1.063. and HDL Ž1.063 - d 1.21. were isolated by discontinuous density gradient ultracentrifugation, as described by Redgrave et al. w16x and modified by Terpstra et al. w17x. Total lipids were extracted from liver samples by the method of Folch et al. w18x. Total and esterified cholesterol was measured in the extracts, using CHOD-PAP and FCHOL kits. Liver triacylglycerol content was also determined using GPO-PAP kit from Randox Laboratories Canada ŽMississauga, Ontario, Canada. . Nitrite levels were measured in serum samples using a nitraternitrite assay kit ŽCayman Chemical, Ann Arbor, MI, USA. . Before the determination, samples were diluted with nitraternitrite-free distilled water and ultrafiltered for 120 min at 2000 = g ŽUltrafree MC microfuge device, Millipore, Bedford, MA, USA. w19x. 2.2. Cell culture studies Minimum essential medium ŽMEM. , fetal bovine serum ŽFBS. and fatty acid-free bovine serum albumin ŽBSA. fraction V were obtained from Life Technologies ŽBurlington, Ontario, Canada. . SNAP, N– OH–Arg, L-arginine and its analogues, L-NMA and D-NMA, were purchased from Sigma Ž St. Louis, MO, USA.. The radiolabeled w4,5- 3 Hx-leucine was received from ICN Pharmaceuticals, Irvine, CA, USA. and the radiolabeled 1- 14 C-acetate was from Amersham ŽOakville, ON, Canada. . The human hepatoma cell line, HepG2, was obtained from the American Type Culture Collection Ž Rockville, MD, USA. . Cells were grown and maintained in 80 cm2 flasks at 378C in a humidified atmosphere of 95% air-5% CO 2 in MEM containing 10% FBS and 100 IU penicillinrstreptomycin. Flasks were subcultured at 1:4 ratio every 7 days, using 0.25% trypsin in Ca2q and Mg 2q-free phosphate-buffered saline Ž PBS. . For experiments, cells were seeded in 24-well or 6-well plates Ž6 = 10 5 cellsrplate. and used at confluence. Before experiments, cells were preincubated for 24 h with MEM containing 1% BSA instead of FBS. They were subsequently incubated for 24 h in MEMrBSA medium supplemented with freshly prepared SNAP, L-arginine or N–OH–Arg provided at indicated nontoxic concentrations, as determined by the MTT viability assay w20x. In all experiments
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except those testing responses to L-arginine, the control media contained MEM and BSA without supplements. When effects of L-arginine were tested with or without its analogues, L-NMA and D-NMA, control media were supplemented with a mixture of L-serine, L-proline and L-aspartic acid, at the comparable level Ž5 mgrl.. This particular combination of nonessential amino acids had no effect on the apo B content of the media in our previous studies w13x. After incubation, media were collected and apo B concentrations were measured as in our earlier experiments w13x, using an enzyme-linked immunosorbent assay ŽELISA. described by Young et al. w21x and modified by Ortho Diagnostics ŽLaJolla, CA, USA. . In parallel, effect of SNAP on apo B concentration was determined using a control, apo B-containing medium from HepG2 cells incubated in absence of SNAP for 24 h. This was done to exclude a possibility of loss of apo B reactivity in the assay in presence of NO donor. Cells were subsequently washed 3 times with cold PBS, cellular proteins were extracted with 0.1 N NaOH and quantitated with the Coomassie Plus Protein Assay ŽPierce, Rockford, IL, USA. . In some studies, medium levels of NO end products, nitrate and nitrite, were measured, using nitraternitrite assay kit ŽCayman Chemical.. To determine whether incubation with SNAP could affect total cellular synthesis and secretion of protein, confluent cells were washed with Hanks’ balanced salt solution and then incubated with control or experimental media in presence of 3 H-leucine Ž5 dpmrpmol. for 0–120 min. The media and cells were collected at the indicated time points and aliquots were applied to 20 mm cellulose acetate filters. Proteins were precipitated with 10% trichloroacetic acid and washed from excess label, as described by Everson et al. w22x. Protein precipitates were solubilized in a mixture of 0.1 N NaOH and Scintigest Ž Fisher Scientific, Fair Lawn, NY, USA. and incorporation of radioactivity into intracellular and secreted protein was determined. To determine whether exposure to SNAP altered intracellular cholesterol metabolism, confluent HepG2 cells were incubated for 24 h in MEMrBSA with or without 0.5 mM SNAP in the presence of 1- 14 Cacetate Ž0.5 m Cirml medium.. Cells were washed three times with ice-cold phosphate-buffered saline and radiolabeled lipids were extracted using hep-
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E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
tanerisopropyl alcohol Ž 3:2, vrv.. Free cholesterol, cholesterol esters and triacylglycerols were separated by thin-layer chromatography, using a hexanerethyl etherracetic acid Ž75:25:1, vrv. solvent system. The lipid fractions were scraped into scintillation vials and radioactivity was determined. Each point in each experiment is the average of triplicate determinations. Significance was measured by paired Student’s t-test.
Table 2 Effects of dietary NaNP supplementation on CErFC ratio in serum lipoprotein fractions Diet
VLDL
LDL
HDL
Control 0.03% NaNP
5.3"0.6 5.9"0.3
4.8"0.4 5.6"0.3
3.7"0.8 7.3"1.1)
Means"SEM of five to six rabbits per group. )Significantly different from control by Student t-test P - 0.02.
3. Results
the LDL and VLDL fractions. Treatment with NaNP did not have significant effect on liver lipids or on the CE:FC ratio in the liver Žnot shown. .
3.1. Animal studies
3.2. Cell culture studies
Growth performance, serum lipid profiles and serum nitrite concentrations in rabbits fed the casein diet with and without supplementation of NaNP are presented in Table 1. The results showed that after 4 weeks of feeding, animals in both groups had similar weight gains, food consumption, serum total cholesterol, VLDL and HDL cholesterol as well as total triacylglycerols. However, treatment with NaNP caused a significant 36% reduction of LDL cholesterol. There was no significant difference between the serum content of nitrite of the two groups. Changes in cholesterol esters:free cholesterol Ž CE:FC. ratio in lipoprotein fractions from rabbits fed control vs. experimental diet are presented in Table 2. These results show that dietary supplementation with NaNP significantly increased the relative proportion of CE in HDL but only tended to increase this proportion in
Effects of exposure of HepG2 cells to increasing, nontoxic doses of SNAP on net apo B production and on medium content of nitratesrnitrites are presented in Figs. 1 and 2. Addition of increasing levels of SNAP to a standard MEM medium caused a dose-dependent reduction of apo B ŽFig. 1. and a simultaneous dose-dependent increase of nitratesrnitrites ŽFig. 2. in the medium of HepG2 cells after 24 h incubation. In comparison, no apo B reduction was observed after addition of SNAP to control medium containing apo B previously secreted in its absence.
Table 1 Effects of NaNP supplementation on growth performance, serum lipids and serum nitrite in rabbits fed casein diet for 4 weeks Group
Control
0.03% NaNP
Initial weight, kg Weight gain, grday Food consumption, grday Total cholesterol, mmolrl VLDL cholesterol, mmolrl LDL cholesterol, mmolrl HDL cholesterol, mmolrl Triacylglycerols, mmolrl Serum nitrite, m molrl
1.81"0.05 19"4 75"9 9.3"0.7 1.5"0.6 6.1"0.6 0.6"0.3 0.8"0.2 16.4"1.2
1.83"0.03 23"3 84"6 6.4"1.3 1.4"0.2 3.9"0.8) 0.7"0.1 0.9"0.2 20.8"6.0
Means"SEM of five to six rabbits per group. )Significantly different from control by Student t-test, P - 0.05.
Fig. 1. Effect of varying levels of nitric oxide donor, SNAP, on apo B content in medium of HepG2 cells. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA with indicated concentrations of freshly prepared SNAP. Apoprotein concentrations were measured by immunoassay and are expressed as micrograms of apo Brmilligram cell protein"SEM for an average of 3 experiments. ) P - 0.05.
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
Fig. 2. Concentrations of nitratesqnitrites in experimental media. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA with indicated concentrations of freshly prepared SNAP. Nitrates q nitrites concentration was measured using nitraternitrite assay kit and expressed as micrograms of nitrates qnitritesrmilligram cell protein"SEM for an average of 3 experiments.
Figs. 3 and 4 demonstrate changes with time in total cellular synthesis and secretion of proteins in the presence and absence of apo B-reducing concentra-
Fig. 3. Effect of 0.5 mM SNAP on total cellular protein synthesis. HepG2 cells were incubated for 20–120 min in serum-free medium with 1% BSA and either with Ž — — . or without Ž- - -. 0.5 mM SNAP. Total radiolabeled intracellular proteins were precipitated with trichloroacetic acid and counted. Results are expressed as mean nanomoles of w 3 Hx-leucine incorporated into trichloroacetic acid-precipitable proteinrmilligram of cell protein "SEM for an average of 3 experiments.
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Fig. 4. Effect of 0.5 mM SNAP on total secreted protein synthesis. HepG2 cells were incubated for 20–120 min in serum-free medium with 1% BSA and either with Ž — — . or without Ž- - -. 0.5 mM SNAP. Total radiolabeled proteins from the medium were precipitated with trichloroacetic acid and counted. Results are expressed as mean nanomoles of w 3 Hx-leucine incorporated into trichloroacetic acid-precipitable proteinrmilligram of cell protein"SEM for an average of 3 experiments.
tion of SNAP Ž 0.5 mM. . These results show that the amounts of radiolabeled proteins synthesized Ž Fig. 3. and secreted ŽFig. 4. by HepG2 cells were not altered by SNAP. To determine if changes in intracellular metabolism of lipids, especially cholesterol and cholesterol esters, were responsible for the alteration in apo B metabolism, incorporation of 14C-acetate into cellular lipids was measured in cells incubated in the presence or absence of 0.5 mM SNAP. As shown in Fig. 5, there was no evidence of any influence of SNAP on intracellular metabolism of lipids. To assess whether increases in NO synthesis could be responsible for reduction of medium apo B in HepG2 cells exposed to high exogenous levels of L-arginine in our previous studies w13x, we tested whether the NO synthase inhibitor, L-NMA, as well as its inactive version, D-NMA, can counteract the changes in apo B caused by L-arginine. The results presented in Fig. 6 show that the effect of L-arginine was reversed by both L-NMA and D-NMA. Another experiment was done to determine whether incubation of HepG2 cells with L-arginine can stimulate synthesis of NO by measuring the accumulation of nitratesrnitrites in cell culture after 24 h. The results demonstrated that the culture medium contained in-
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E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
Fig. 5. Effects of 0.5 mM SNAP on rates of incorporation of radiolabeled acetate into intracellular lipids. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA and either with or without 0.5 mM SNAP, in the presence of w 14 Cxacetate Ž0.5 m Cirml.. Radioactivity incorporated into cellular free cholesterol, cholesterol esters and triacylglycerols was measured after extraction of cellular lipids and their separation by thin-layer chromatography. Results were expressed as nanomoles of w 14 Cx-acetate incorporated per milligram of cell protein"SEM. Average of 4 experiments.
Fig. 6. Reversal of the apo B-reducing effect of L-arginine by 2 mM L-NMA and D-NMA. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA supplemented with 1 grl of L-arginine and 4 grl of L-serinerL-prolinerL-aspartic acid Ž12:13:15, vrv. Žgrey bars.. The indicated samples contained additionally 2 mM of L-NMA or D-NMA Žhatched bars.. The control medium Žwhite bar. was supplemented with 5 grl of L-serinerL-prolinerL-aspartic acid Ž12:13:15, vrv.. Apoprotein concentrations were measured by immunoassay and are expressed as percent of control"SEM for an average of 3 experiments. ) P - 0.05.
different, trace amounts of NO metabolites after incubation of HepG2 cells either with or without high levels of L-arginine Žnot shown.. Because it was clear from the experiment above that in HepG2 cells, incubation with excess of Larginine reduced medium content of apo B without stimulating synthesis of NO, we subsequently investigated whether a similar reduction of medium apo B but possibly associated with increased release of NO could be induced by exposure of these cells to nontoxic concentrations of a stable intermediate product of L-arginine, N–OH–Arg. Our results demonstrated that incubation of HepG2 cells with increasing concentrations of N–OH–Arg Ž0.1–1.0 mM. did not alter apo B concentrations and did not increase the low, basal levels of nitratesrnitrites in cell culture medium.
4. Discussion Our experiment demonstrated for the first time that in rabbits fed cholesterol-free, semipurified diet containing casein, chronic oral administration of the NO-generating compound, NaNP, counteracted the elevation of LDL cholesterol induced by the diet but did not affect VLDL and HDL cholesterol. This observation is consistent with previous reports showing that increasing activity of NO lowers serum cholesterol whereas decreasing it exacerbates hypercholesterolemia w2–4x. Reduction of LDL cholesterol induced by dietary NaNP was not associated with any toxic or adverse effects in experimental animals, as confirmed by lack of changes in food consumption and body weight. Daily intake of NaNP also did not appear to reduce blood pressure in experimental animals after 4 weeks of treatment, as measured in two control vs. two experimental rabbits Ž unpublished data.. Our results suggested therefore that dietary administration of NaNP affected mainly LDL metabolism and that NaNP could act either by reducing de novo synthesisrsecretion of LDL particles andror by increasing their catabolism. The hypocholesterolemic responses obtained in our study were relatively marked and comparable to the responses induced by dietary NaNP in Japanese quail fed cholesterol-enriched diet w4x. In contrast, much less pronounced increases of serum cholesterol were
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
reported earlier in experiments using NO-lowering agents w2,3x. This difference in effectiveness between dietary NO donor and inhibitors could be related to the fact that the donor was able to counteract the NO depletion caused by hypercholesterolemia. In contrast, NO-lowering agents had limited capability to act since in animals with hypercholesterolemia, blood levels of the endogenous inhibitor of NO synthase, ADMA, have been reported to be already elevated w6,7x. The hypocholesterolemia induced in rabbits by treatment with NaNP apparently differed from the acquired hypocholesterolemia observed during inflammation and tissue injury. Thus, both NaNP and inflammation caused a reduction of LDL cholesterol, but inflammation also increased plasma triacylglycerols and decreased HDL cholesterol in many species, including the rabbit w23,24x. These latter effects were likely due to release of cytokines associated with the inflammatory state w8x. Another NaNPinduced change in lipoprotein metabolism reported in our study, cholesterol ester enrichment of HDL, could be related to a specific effect of NaNP itself. Cholesterol ester ŽCE. enrichment of lipoproteins, especially VLDL and LDL, has usually been linked to accumulation of CE in the liver and was typically observed during hypercholesterolemia w25x. In contrast, dietary NaNP increased CE mainly in the HDL fraction and this occurred without altering CE and other lipids in the liver. Our in vitro studies show clearly that exposure of HepG2 cells to increasing, nontoxic concentrations of SNAP caused a dose-dependent reduction of medium level of apo B, the main protein component of LDL. This response corresponded to the LDL cholesterollowering effect of dietary NaNP, suggesting that regulation of lipoprotein metabolism by the NO-generating compounds in vivo could occur directly in the liver. The simultaneous dose-dependent increases in medium concentrations of NO end metabolites, nitrate and nitrite, indicated that in HepG2 cells, exogenous NO could be directly involved in inducing hypocholesterolemic effects. A direct role of endogenous NO in regulation of cholesterolemic responses has also been postulated in recent studies which showed that in rats treated with an NO synthase inhibitor, L-NNA, elevation of serum cholesterol was associated with reduced postprandial levels of serum
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nitrites w3x. In our in vivo study, on the other hand, serum nitrites were not significantly increased in rabbits given NaNP, possibly because in blood samples collected from the animals 16–18 h after food withdrawal serum nitrites were already normalized. The mechanism by which SNAP suppressed overall apo B production in HepG2 cells has not been yet completely elucidated. Our study demonstrated that at the concentration of 0.5 mM, SNAP significantly decreased medium apo B but did not reduce total synthesis and secretion of cellular proteins. In agreement, incubation with SNAP at a similar concentration did not cause any reduction of total cellular protein synthesis in primary rat hepatocytes w26x. The effect of SNAP on apo B also appeared to be independent of intracellular synthesis of lipids, since SNAP at a concentration of 0.5 mM did not alter rates of 14C-acetate incorporation into cellular cholesterol, CE and triacylglycerols. This was consistent with the lack of changes in liver lipids in rabbits given NaNP, implying a similarity between mechanisms of action of NO donors in vivo and in vitro. In contrast, incubation of HepG2 cells with cytokines has been shown to increase rates of 14C-acetate incorporation into cellular sterols w8x, suggesting a difference between the hypocholesterolemic effects induced by NO donors and by inflammation. Overall, our data support the hypothesis that in HepG2 cells, SNAP suppressed net apo B production via an apo B-specific, cellular sterol-independent mechanism. However, it is still not clear whether changes in the medium level of apo B were caused by its decreased synthesisrsecretion or by increased catabolism of apo B-containing lipoproteins. Our finding that drugs that release NO can produce hypocholesterolemic responses in rabbits as well as in HepG2 cells suggested that physiological donors of NO could contribute to the maintenance of normal metabolism of lipoproteins or possibly play a role in counteracting hypercholesterolemia. However, little evidence has been found so far to support the hypothesis that exogenous L-arginine, which is a natural precursor in NO synthesis, could influence cholesterolemic responses by enhancing synthesis of NO. Our present results show that in HepG2 cells exposed to L-arginine, addition of an L-arginine analogue and NO synthase inhibitor, L-NMA, or its non-active isoform, D-NMA, to the culture medium resulted in
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E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
equally effective reversal of apo B reduction induced by this amino acid. This suggested that both compounds counteracted the hypocholesterolemic effect of L-arginine by competing with it for transport sites across the hepatocyte plasma membrane w27x rather than by preventing its conversion to NO. In agreement with this observation, decreases in medium apo B induced by exposure of HepG2 cells to high exogenous levels of L-arginine were not associated with increased medium levels of nitratesrnitrites, confirming the absence of NO synthase activity in this cell line. The lack of involvement of NO in regulation of cholesterolemia by exogenous L-arginine has also been suggested by results in vivo. In our previous rabbit experiments, addition of L-arginine to hypercholesterolemic, amino acid-based diets reduced LDL cholesterol w28x. However, the response developed earlier than in animals given NaNP Ž after 2–3 weeks. and no associated increases were observed in plasma or liver content of NO metabolites Ž unpublished data. . In contrast, a number of other studies using proteinbased diets showed that supplementary L-arginine improved the vascular tone andror inhibited atherogenesis, largely due to its metabolism to NO w9x, but failed to reduce hypercholesterolemia w5,10,11x. This lack of cholesterolemic activity could be at least partially related to the fact that single amino acids added as supplements to protein-based diets usually have limited influence on cholesterolemia w29x. Additionally, constitutive NO synthase has been known to be poorly stimulated by physiological concentrations of L-arginine, which are much higher than its K m for this amino acid w30x. Another possibility, that a stable intermediate in NO biosynthesis from L-arginine, N–OH–Arg, could play a role in regulation of lipoprotein metabolism in the liver, merits further investigation. Previous studies documented that exogenous N–OH–Arg can enter into cultured vascular smooth muscle cells Ž which do not express a constitutive form of NO synthase. and that it can be there oxidized to NO, most likely by cytochrome P450 w14x. We therefore postulated that N–OH–Arg produced in the endothelium of liver vasculature could also diffuse into hepatocytes and there be converted into NO by a similar, constitutive NO synthase-independent mechanism. In contrast to this hypothesis, exposure of HepG2 cells to N–OH– Arg did not reduce medium levels of apo B and also
did not increase the medium content of nitratesrnitrites, suggesting that N–OH–Arg cannot be utilized as a source of NO for regulation of lipoprotein metabolism in the hepatocytes. These results should, however, be verified in vivo, since some of the metabolic responses in HepG2 cells are known to be unique for this cell line w31x, and this could include lack of enzymesrcofactors that convert N– OH–Arg into NO via the NO synthase-independent pathway.
5. Conclusion In summary, our results demonstrated that hypercholesterolemia associated with elevation of LDL cholesterol can be reduced by chronic oral administration of synthetic NO donor. However, our data did not provide evidence that a similar effect could be induced by replacing these synthetic donors with supplementary levels of a physiological NO precursor, L-arginine. The NO-generating drugs appear to affect metabolism of lipoproteins directly in the liver and most likely act by counteracting the depletion of NO caused by hypercholesterolemia, via a cellular sterol-independent mechanism. Future studies should be done to investigate in more detail how NO donors interact with metabolism of lipoproteins in the liver. These studies may lead to new therapeutic approaches aimed at reversing hypercholesterolemia and slowing the progression of cardiovascular disease.
Acknowledgements Supported by the Heart and Stroke Foundation of Ontario. The authors wish to thank Ms. Mary Moffatt, Ms. Trang Thu Tran and Ms. Debbie Friedrich for their expert technical assistance. We also extend our thanks to Dr. David Topping for his comments on the manuscript.
References w1x W. Jessup, Oxidized lipoproteins and nitric oxide, Curr. Opin. Lipidology 7 Ž1996. 247–280.
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50 w2x K. Naruse, K. Shimizu, M. Muramatsu, Y. Toki, Y. Miyzaki, K. Okumura, H. Hashimoto, T. Ito, Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta. PGH 2 does not contribute to impaired endothelium-dependent relaxation, Arterioscler. Thromb. 14 Ž1994. 746–752. w3x A. Khedara, Y. Kawai, J. Kayashita, N. Kato, Feeding rats the nitric oxide synthase inhibitor, L-N v nitroarginine, elevates serum triglyceride and cholesterol and lowers hepatic fatty acid oxidation, J. Nutr. 126 Ž1996. 2563–2567. w4x C.H. Hill, E.P. Pullman, B. Starcher, J.C. Shih, Dietary nitroprusside alleviates atherosclerosis in hypercholesterolemic Japanese quail Ž Coturnix coturnix japonica., Comp. Biochem. Physiol. A Physiol. 112 Ž1995. 151–154. w5x R.H. Boger, S.M. Bode-Boger, A. Muge, S. Kienke, R. ¨ ¨ ¨ Brandes, A. Dwenger, J.C. Frolich, Supplementation of ¨ hypocholesterolaemic rabbits with L-arginine reduces the vascular release of superoxide anions and restores NO production, Atherosclerosis 117 Ž1995. 273–284. w6x X. Yu, Y. Li, Y. Xiong, Increase of an endogenous inhibitor of nitric oxide synthase in serum of high cholesterol fed rabbits, Life Sci. 54 Ž1994. 753–758. w7x S.M. Bode-Boger, R.H. Boger, S. Kienke, W. Junker, J.C. ¨ ¨ Frolich, Elevated L-argininerdimethylarginine ratio con¨ tributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits, Biochem. Biophys. Res. Commun. 219 Ž1996. 598–603. w8x W.H. Ettinger, V.K. Varma, M. Sorci-Thomas, J.S. Parks, R.C. Sigmon, T.K. Smith, R.B. Verdery, Cytokines decrease apolipoprotein accumulation in medium from HepG2 cells, Arterioscler. Thromb. 14 Ž1994. 8–13. w9x J.P. Cooke, P.S. Tsao, Arginine: a new therapy for atherosclerosis?, Circulation 95 Ž1997. 311–312. w10x J.P. Cooke, A.H. Singer, P. Tsao, P. Zera, R.A. Rowan, M.E. Billingham, Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit, J. Clin. Invest. 90 Ž1992. 1168–1172. w11x W. Aji, S. Ravalli, M. Szaboles, X. Jiang, R.R. Sciacca, R.E. Michler, P.J. Cannon, L-arginine prevents xanthoma development and inhibits atherosclerosis in LDL receptor knockout mice, Circulation 95 Ž1997. 430–437. w12x E.M. Kurowska, K.K. Carroll, Hypercholesterolemic responses in rabbits to selected groups of dietary essential amino acids, J. Nutr. 124 Ž1994. 364–370. w13x E.M. Kurowska, K.K. Carroll, LDL versus apolipoprotein B responses to variable proportions of selected amino acids in semipurified diets to rabbits and in the media of HepG2 cells, J. Nutr. Biochem. 7 Ž1996. 418–424. w14x C.A. Schott, C.M. Bogen, P. Vetrovsky, C.C. Berton, J.C. Stoclet, Exogenous N G-hydroxy-L-arginine causes nitrite production in vascular smooth muscle cells in the absence of nitric oxide synthase activity, FEBS Lett. 341 Ž1994. 203– 207. w15x J.M. Hrabek-Smith, E.M. Kurowska, K.K. Carroll, Effects of cholesterol-free, semipurified diets containing different levels of casein or soy protein on distribution of cholesterol
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and protein among serum lipoproteins of rabbits, Atherosclerosis 76 Ž1989. 125–130. T.G. Redgrave, D.C.K. Roberts, C.E. West, Separation of plasma lipoproteins by density gradient ultracentrifugation, Anal. Biochem. 63 Ž1975. 42–49. A.H.M. Terpstra, C.J.H. Woodward, F.J. Sanchez-Muniz, Improved techniques for the separation of serum lipoproteins by density gradient ultracentrifugation: visualization by prestaining and rapid separation of serum lipoproteins from small volumes of serum, Anal. Biochem. 111 Ž1981. 149– 157. J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 Ž1957. 497–509. D.L. Granger, R.R. Taintor, K.S. Boockvar, J.B. Hibbs Jr., Determination of nitrate and nitrite in biological samples using bacterial nitrate reductase coupled with the Griess reaction, Methods Enzymol. 7 Ž1995. 78–83. M.B. Hansen, S.E. Nielsen, K. Berg, Reexamination and further development of a precise and rapid dye method for measuring cell growthrcell kill, J. Immunol. Methods 119 Ž1989. 203–210. S. Young, R.S. Smith, D. Hogle, L. Curtiss, J. Witzum, Two new monoclonal antibody-based enzyme assays of apolipoprotein B, Clin. Chem. 32 Ž1986. 1484–1490. W.V. Everson, K.E. Flaim, D.M. Susco, S.R. Kimball, L.S. Jefferson, Effect of amino acid deprivation on initiation of protein synthesis in rat hepatocytes, Am. J. Physiol. 256 Ž1989. C18–C27. W.H. Ettinger, L.D. Miller, J.J. Albers, T.K. Smith, J.S. Parks, Lipopolysaccharide and tumor necrosis factor cause a fall in plasma concentration of lecithin: cholesterol acyltransferase in cynomolgus monkeys, J. Lipid Res. 31 Ž1990. 1099–1107. V.G. Cabana, J.N. Siegel, S.M. Sabesin, Effects of the acute phase response on the concentration and density distribution of plasma lipids and apolipoproteins, J. Lipid Res. 30 Ž1989. 39–49. K.E. Suckling, E.F. Stange, Role of acyl-CoA: cholesterol acyltransferase in cellular cholesterol metabolism, J. Lipid Res. 26 Ž1985. 647–671. R.D. Curran, F.K. Ferrari, P.H. Kispert, J. Stadler, D.J. Stuehr, R.L. Simmons, T.R. Billiar, Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis, FASEB J. 5 Ž1991. 2085–2092. Y. Inoue, B.P. Bode, D.J. Beck, A.P. Li, K.I. Bland, W.W. Souba, Arginine transport in human liver: characterization and effects of nitric oxide synthase inhibitors, Ann. Surg. 218 Ž1993. 350–363. E.M. Kurowska, K.K. Carroll, Effect of high levels of selected dietary essential amino acids on hypocholesterolemia and down-regulation of hepatic LDL receptors in rabbits, Biochem. Biophys. Acta 1126 Ž1992. 185–191. A.H.M. Terpstra, R.J.J. Hermus, C.E. West, Dietary protein and cholesterol metabolism in rabbits and rats, in: M.J. Gibney, D. Kritchevsky, ŽEds.., Animal and Vegetable Pro-
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teins in Lipid Metabolism and Atherosclerosis, Alan R. Liss, New York, 1983, pp. 19-49. w30x U. Forstermann, E.I. Closs, J.S. Pollock, M. Nakane, P. Schwarz, I. Gath, H. Kleinert, Nitric oxide synthase isoenzymes. Characterization, purification, molecular cloning, and functions, Hypertension 23 Ž1994. 1121–1131.
w31x R.N. Thrift, T.M. Forte, B.E. Cahoon, V.G. Shore, Characterization of lipoproteins produced by the human liver cell line, HepG2, under defined conditions, J. Lipid Res. 27 Ž1986. 236–250.
Hypocholesterolemic properties of nitric oxide. In vivo and in vitro studies using nitric oxide donors E.M. Kurowska ) , K.K. Carroll Department of Biochemistry, Centre for Human Nutrition, UniÕersity of Western Ontario, London, Ontario, Canada, N6A 5C1 Received 18 August 1997; revised 17 December 1997; accepted 19 December 1997
Abstract Previous results suggested that changes in the activity of nitric oxide ŽNO. can influence metabolism of apo B-containing lipoproteins. Therefore, we studied effects of exogenous NO donors and physiological NO precursors on metabolism of these lipoproteins. In rabbits, addition of 0.03% sodium nitroprusside ŽNaNP. to a semipurified, cholesterol-free, casein diet counteracted the elevation of LDL cholesterol induced by this diet but did not alter liver lipids after 4 weeks of feeding. In HepG2 cells, addition of nontoxic concentrations of another NO donor, S-nitroso-N-acetylpenicillamine ŽSNAP. to culture medium caused a dose-dependent reduction of medium apo B after 24 h. At the concentration 0.5 mM, SNAP significantly decreased medium apo B by 50% without altering total synthesis and secretion of proteins and without altering rates of cellular sterol synthesis. In cells incubated with L-arginine, reduction of medium apo B was not associated with increased NO production whereas in those exposed to N–OH–Arg medium apo B levels were not altered. We concluded that synthetic NO donors can reduce hypercholesterolemia by affecting apo B metabolism directly in the liver, via the sterol-independent mechanism. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide donor; Cholesterol; HepG2 cell; ŽRabbit.
1. Introduction There is a growing body of evidence that nitric oxide ŽNO., the endothelium-derived relaxing factor originating from metabolism of L-arginine, can prevent atherosclerosis by inhibiting oxidation of lipoproteins in the arterial wall w1x. In addition, some recent studies have suggested that NO can modulate metabolism of lipoproteins when its availability is altered by administration of synthetic NO synthase inhibitors or NO donors. In cholesterol-fed rabbits, )
Corresponding author. Fax: q1-519-661-4006; E-mail: [email protected]
chronic administration of the NO synthase inhibitor, N v-nitro-L-arginine ŽL-NAME., promoted atherosclerosis and also tended to increase hypercholesterolemia w2x. Similarly, serum cholesterol level was moderately elevated in rats treated with another NO synthase inhibitor, L-N v Nitroarginine ŽL-NNA. w3x. Conversely, a substantial, dose-dependent reduction of hypercholesterolemia as well as atherosclerosis was observed in cholesterol-fed Japanese quail after chronic oral administration of an NO donor, sodium nitroprusside ŽNaNP. w4x. A relationship between activity of NO and cholesterolemia has also been found in the absence of pharmacological intervention. In rabbits with diet-in-
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 2 1 5 - 4
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E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
duced hypercholesterolemia, urinary excretion of NO metabolic products, nitrites, was decreased w5x and plasma levels of an endogenous NO synthase inhibitor, N G, N G-dimethylarginine, were increased, suggesting impaired NO activity w6,7x. In agreement, an excessive release of NO during inflammation and tissue injury has been associated with acquired hypocholesterolemia, although this response has been postulated to be at least partly due to preceding release of cytokines w8x. Previous results suggested that NO-mediated hypocholesterolemic responses are unlikely to be enhanced by increased dietary intake of NO precursor, L-arginine. Dietary supplementation with L-arginine has been shown to reverse endothelial dysfunction caused by hypercholesterolemia and to inhibit atherogenesis w9x but failed to counteract hypercholesterolemia itself w5,10,11x, except when the endogenous pool of L-arginine was severely depleted by prior chronic administration of NO inhibitors w3x. In contrast, in our recent studies, dietary L-arginine appeared to have anti-hypercholesterolemic properties in rabbits fed hypercholesterolemic amino acid diets as well as apo B-lowering properties in HepG2 cells w12,13x. However, in the rabbit model, feeding high levels of L-arginine was not associated with increased plasma and liver content of NO end metabolites, nitrates Ž unpublished data. . Another possibility, that a stable intermediate precursor in L-arginine-NO pathway, N G-hydroxy-L-arginine ŽN–OH–Arg. , could be important in NO-mediated regulation of lipoprotein metabolism, is yet to be investigated. It has been shown that in vitro, N–OH–Arg can be oxidized to NO and nitrite in the absence of active NO synthase, probably by cytochrome P450 w14x. The mechanism by which changes in activity of NO can influence the metabolism of lipoproteins is poorly understood. The lipoprotein responses produced in animals treated with inhibitors of NO synthase, although obscured by cholesterol feeding, suggested that VLDL andror LDL rather than HDL cholesterol are likely to be affected w2x. Since VLDL and LDL are synthesized and catabolized in the liver, we hypothesized that regulation of lipoprotein metabolism by NO, directly or via its intermediate precursors, L-arginine or N–OH–Arg, could occur in this organ. To understand the nature of this regulation better, we investigated effects of exogenous NO
donors on metabolism of lipoproteins in vivo and in vitro. In vivo, effects of 0.03% NaNP supplementation on lipoprotein profile and on liver lipids was tested using rabbits in which experimental hypercholesterolemia associated with elevation of LDL cholesterol, similar to that in humans, was induced by feeding a semipurified, cholesterol-free, casein diet w15x. In vitro, cholesterolemic responses were analyzed in human hepatoma ŽHepG2. cells after their exposure to a less toxic NO donor, S-nitroso-Nacetylpenicillamine ŽSNAP.. Experiments were also conducted to determine whether in HepG2 cells, the apo B-lowering effect induced by incubation with high levels of L-arginine could be mediated by NO and whether a similar reduction of apo B in the medium could be obtained by exposure of HepG2 cells to N-OH-Arg.
2. Materials and methods 2.1. Animal experiment The animal protocol was in accordance with the Canadian Council of Animal Care guidelines and was approved by the Council of Animal Care, University of Western Ontario. Young New Zealand White male rabbits ŽReimen’s Fur Ranches, Guelph, Ontario, Canada., weighing 1.6–1.7 kg, were used in these experiments. The animals were housed individually in galvanized cages with wire bottoms, in a room maintained at 21–248C with a 12-h light:dark cycle. Following adaptation period w15x, they were randomly divided into two groups, 6 animals each, and given low-fat, cholesterol-free, semipurified diet containing 25% casein, with or without addition of 0.03% NaNP, for a period of 4 weeks. The composition of the casein diet was described previously w15x. Food and water were provided ad libitum. Weight gains and food consumption were monitored weekly. At the end of the study, food was withdrawn, the animals were killed 16–18 h later by an overdose of Euthanyl Forte ŽCanada Packers, Cambridge, Ontario, Canada. and blood samples were taken by heart puncture. Total and free cholesterol in serum and lipoprotein fractions were measured with enzymatic kits ŽCHOD-PAP and F-CHOL, Boehringer-Man-
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
nheim, Montreal, Canada.. VLDL Ž d - 1.006 grml., LDL Ž 1.006 - d - 1.063. and HDL Ž1.063 - d 1.21. were isolated by discontinuous density gradient ultracentrifugation, as described by Redgrave et al. w16x and modified by Terpstra et al. w17x. Total lipids were extracted from liver samples by the method of Folch et al. w18x. Total and esterified cholesterol was measured in the extracts, using CHOD-PAP and FCHOL kits. Liver triacylglycerol content was also determined using GPO-PAP kit from Randox Laboratories Canada ŽMississauga, Ontario, Canada. . Nitrite levels were measured in serum samples using a nitraternitrite assay kit ŽCayman Chemical, Ann Arbor, MI, USA. . Before the determination, samples were diluted with nitraternitrite-free distilled water and ultrafiltered for 120 min at 2000 = g ŽUltrafree MC microfuge device, Millipore, Bedford, MA, USA. w19x. 2.2. Cell culture studies Minimum essential medium ŽMEM. , fetal bovine serum ŽFBS. and fatty acid-free bovine serum albumin ŽBSA. fraction V were obtained from Life Technologies ŽBurlington, Ontario, Canada. . SNAP, N– OH–Arg, L-arginine and its analogues, L-NMA and D-NMA, were purchased from Sigma Ž St. Louis, MO, USA.. The radiolabeled w4,5- 3 Hx-leucine was received from ICN Pharmaceuticals, Irvine, CA, USA. and the radiolabeled 1- 14 C-acetate was from Amersham ŽOakville, ON, Canada. . The human hepatoma cell line, HepG2, was obtained from the American Type Culture Collection Ž Rockville, MD, USA. . Cells were grown and maintained in 80 cm2 flasks at 378C in a humidified atmosphere of 95% air-5% CO 2 in MEM containing 10% FBS and 100 IU penicillinrstreptomycin. Flasks were subcultured at 1:4 ratio every 7 days, using 0.25% trypsin in Ca2q and Mg 2q-free phosphate-buffered saline Ž PBS. . For experiments, cells were seeded in 24-well or 6-well plates Ž6 = 10 5 cellsrplate. and used at confluence. Before experiments, cells were preincubated for 24 h with MEM containing 1% BSA instead of FBS. They were subsequently incubated for 24 h in MEMrBSA medium supplemented with freshly prepared SNAP, L-arginine or N–OH–Arg provided at indicated nontoxic concentrations, as determined by the MTT viability assay w20x. In all experiments
43
except those testing responses to L-arginine, the control media contained MEM and BSA without supplements. When effects of L-arginine were tested with or without its analogues, L-NMA and D-NMA, control media were supplemented with a mixture of L-serine, L-proline and L-aspartic acid, at the comparable level Ž5 mgrl.. This particular combination of nonessential amino acids had no effect on the apo B content of the media in our previous studies w13x. After incubation, media were collected and apo B concentrations were measured as in our earlier experiments w13x, using an enzyme-linked immunosorbent assay ŽELISA. described by Young et al. w21x and modified by Ortho Diagnostics ŽLaJolla, CA, USA. . In parallel, effect of SNAP on apo B concentration was determined using a control, apo B-containing medium from HepG2 cells incubated in absence of SNAP for 24 h. This was done to exclude a possibility of loss of apo B reactivity in the assay in presence of NO donor. Cells were subsequently washed 3 times with cold PBS, cellular proteins were extracted with 0.1 N NaOH and quantitated with the Coomassie Plus Protein Assay ŽPierce, Rockford, IL, USA. . In some studies, medium levels of NO end products, nitrate and nitrite, were measured, using nitraternitrite assay kit ŽCayman Chemical.. To determine whether incubation with SNAP could affect total cellular synthesis and secretion of protein, confluent cells were washed with Hanks’ balanced salt solution and then incubated with control or experimental media in presence of 3 H-leucine Ž5 dpmrpmol. for 0–120 min. The media and cells were collected at the indicated time points and aliquots were applied to 20 mm cellulose acetate filters. Proteins were precipitated with 10% trichloroacetic acid and washed from excess label, as described by Everson et al. w22x. Protein precipitates were solubilized in a mixture of 0.1 N NaOH and Scintigest Ž Fisher Scientific, Fair Lawn, NY, USA. and incorporation of radioactivity into intracellular and secreted protein was determined. To determine whether exposure to SNAP altered intracellular cholesterol metabolism, confluent HepG2 cells were incubated for 24 h in MEMrBSA with or without 0.5 mM SNAP in the presence of 1- 14 Cacetate Ž0.5 m Cirml medium.. Cells were washed three times with ice-cold phosphate-buffered saline and radiolabeled lipids were extracted using hep-
44
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
tanerisopropyl alcohol Ž 3:2, vrv.. Free cholesterol, cholesterol esters and triacylglycerols were separated by thin-layer chromatography, using a hexanerethyl etherracetic acid Ž75:25:1, vrv. solvent system. The lipid fractions were scraped into scintillation vials and radioactivity was determined. Each point in each experiment is the average of triplicate determinations. Significance was measured by paired Student’s t-test.
Table 2 Effects of dietary NaNP supplementation on CErFC ratio in serum lipoprotein fractions Diet
VLDL
LDL
HDL
Control 0.03% NaNP
5.3"0.6 5.9"0.3
4.8"0.4 5.6"0.3
3.7"0.8 7.3"1.1)
Means"SEM of five to six rabbits per group. )Significantly different from control by Student t-test P - 0.02.
3. Results
the LDL and VLDL fractions. Treatment with NaNP did not have significant effect on liver lipids or on the CE:FC ratio in the liver Žnot shown. .
3.1. Animal studies
3.2. Cell culture studies
Growth performance, serum lipid profiles and serum nitrite concentrations in rabbits fed the casein diet with and without supplementation of NaNP are presented in Table 1. The results showed that after 4 weeks of feeding, animals in both groups had similar weight gains, food consumption, serum total cholesterol, VLDL and HDL cholesterol as well as total triacylglycerols. However, treatment with NaNP caused a significant 36% reduction of LDL cholesterol. There was no significant difference between the serum content of nitrite of the two groups. Changes in cholesterol esters:free cholesterol Ž CE:FC. ratio in lipoprotein fractions from rabbits fed control vs. experimental diet are presented in Table 2. These results show that dietary supplementation with NaNP significantly increased the relative proportion of CE in HDL but only tended to increase this proportion in
Effects of exposure of HepG2 cells to increasing, nontoxic doses of SNAP on net apo B production and on medium content of nitratesrnitrites are presented in Figs. 1 and 2. Addition of increasing levels of SNAP to a standard MEM medium caused a dose-dependent reduction of apo B ŽFig. 1. and a simultaneous dose-dependent increase of nitratesrnitrites ŽFig. 2. in the medium of HepG2 cells after 24 h incubation. In comparison, no apo B reduction was observed after addition of SNAP to control medium containing apo B previously secreted in its absence.
Table 1 Effects of NaNP supplementation on growth performance, serum lipids and serum nitrite in rabbits fed casein diet for 4 weeks Group
Control
0.03% NaNP
Initial weight, kg Weight gain, grday Food consumption, grday Total cholesterol, mmolrl VLDL cholesterol, mmolrl LDL cholesterol, mmolrl HDL cholesterol, mmolrl Triacylglycerols, mmolrl Serum nitrite, m molrl
1.81"0.05 19"4 75"9 9.3"0.7 1.5"0.6 6.1"0.6 0.6"0.3 0.8"0.2 16.4"1.2
1.83"0.03 23"3 84"6 6.4"1.3 1.4"0.2 3.9"0.8) 0.7"0.1 0.9"0.2 20.8"6.0
Means"SEM of five to six rabbits per group. )Significantly different from control by Student t-test, P - 0.05.
Fig. 1. Effect of varying levels of nitric oxide donor, SNAP, on apo B content in medium of HepG2 cells. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA with indicated concentrations of freshly prepared SNAP. Apoprotein concentrations were measured by immunoassay and are expressed as micrograms of apo Brmilligram cell protein"SEM for an average of 3 experiments. ) P - 0.05.
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
Fig. 2. Concentrations of nitratesqnitrites in experimental media. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA with indicated concentrations of freshly prepared SNAP. Nitrates q nitrites concentration was measured using nitraternitrite assay kit and expressed as micrograms of nitrates qnitritesrmilligram cell protein"SEM for an average of 3 experiments.
Figs. 3 and 4 demonstrate changes with time in total cellular synthesis and secretion of proteins in the presence and absence of apo B-reducing concentra-
Fig. 3. Effect of 0.5 mM SNAP on total cellular protein synthesis. HepG2 cells were incubated for 20–120 min in serum-free medium with 1% BSA and either with Ž — — . or without Ž- - -. 0.5 mM SNAP. Total radiolabeled intracellular proteins were precipitated with trichloroacetic acid and counted. Results are expressed as mean nanomoles of w 3 Hx-leucine incorporated into trichloroacetic acid-precipitable proteinrmilligram of cell protein "SEM for an average of 3 experiments.
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Fig. 4. Effect of 0.5 mM SNAP on total secreted protein synthesis. HepG2 cells were incubated for 20–120 min in serum-free medium with 1% BSA and either with Ž — — . or without Ž- - -. 0.5 mM SNAP. Total radiolabeled proteins from the medium were precipitated with trichloroacetic acid and counted. Results are expressed as mean nanomoles of w 3 Hx-leucine incorporated into trichloroacetic acid-precipitable proteinrmilligram of cell protein"SEM for an average of 3 experiments.
tion of SNAP Ž 0.5 mM. . These results show that the amounts of radiolabeled proteins synthesized Ž Fig. 3. and secreted ŽFig. 4. by HepG2 cells were not altered by SNAP. To determine if changes in intracellular metabolism of lipids, especially cholesterol and cholesterol esters, were responsible for the alteration in apo B metabolism, incorporation of 14C-acetate into cellular lipids was measured in cells incubated in the presence or absence of 0.5 mM SNAP. As shown in Fig. 5, there was no evidence of any influence of SNAP on intracellular metabolism of lipids. To assess whether increases in NO synthesis could be responsible for reduction of medium apo B in HepG2 cells exposed to high exogenous levels of L-arginine in our previous studies w13x, we tested whether the NO synthase inhibitor, L-NMA, as well as its inactive version, D-NMA, can counteract the changes in apo B caused by L-arginine. The results presented in Fig. 6 show that the effect of L-arginine was reversed by both L-NMA and D-NMA. Another experiment was done to determine whether incubation of HepG2 cells with L-arginine can stimulate synthesis of NO by measuring the accumulation of nitratesrnitrites in cell culture after 24 h. The results demonstrated that the culture medium contained in-
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E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
Fig. 5. Effects of 0.5 mM SNAP on rates of incorporation of radiolabeled acetate into intracellular lipids. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA and either with or without 0.5 mM SNAP, in the presence of w 14 Cxacetate Ž0.5 m Cirml.. Radioactivity incorporated into cellular free cholesterol, cholesterol esters and triacylglycerols was measured after extraction of cellular lipids and their separation by thin-layer chromatography. Results were expressed as nanomoles of w 14 Cx-acetate incorporated per milligram of cell protein"SEM. Average of 4 experiments.
Fig. 6. Reversal of the apo B-reducing effect of L-arginine by 2 mM L-NMA and D-NMA. HepG2 cells were incubated for 24 h in serum-free medium with 1% BSA supplemented with 1 grl of L-arginine and 4 grl of L-serinerL-prolinerL-aspartic acid Ž12:13:15, vrv. Žgrey bars.. The indicated samples contained additionally 2 mM of L-NMA or D-NMA Žhatched bars.. The control medium Žwhite bar. was supplemented with 5 grl of L-serinerL-prolinerL-aspartic acid Ž12:13:15, vrv.. Apoprotein concentrations were measured by immunoassay and are expressed as percent of control"SEM for an average of 3 experiments. ) P - 0.05.
different, trace amounts of NO metabolites after incubation of HepG2 cells either with or without high levels of L-arginine Žnot shown.. Because it was clear from the experiment above that in HepG2 cells, incubation with excess of Larginine reduced medium content of apo B without stimulating synthesis of NO, we subsequently investigated whether a similar reduction of medium apo B but possibly associated with increased release of NO could be induced by exposure of these cells to nontoxic concentrations of a stable intermediate product of L-arginine, N–OH–Arg. Our results demonstrated that incubation of HepG2 cells with increasing concentrations of N–OH–Arg Ž0.1–1.0 mM. did not alter apo B concentrations and did not increase the low, basal levels of nitratesrnitrites in cell culture medium.
4. Discussion Our experiment demonstrated for the first time that in rabbits fed cholesterol-free, semipurified diet containing casein, chronic oral administration of the NO-generating compound, NaNP, counteracted the elevation of LDL cholesterol induced by the diet but did not affect VLDL and HDL cholesterol. This observation is consistent with previous reports showing that increasing activity of NO lowers serum cholesterol whereas decreasing it exacerbates hypercholesterolemia w2–4x. Reduction of LDL cholesterol induced by dietary NaNP was not associated with any toxic or adverse effects in experimental animals, as confirmed by lack of changes in food consumption and body weight. Daily intake of NaNP also did not appear to reduce blood pressure in experimental animals after 4 weeks of treatment, as measured in two control vs. two experimental rabbits Ž unpublished data.. Our results suggested therefore that dietary administration of NaNP affected mainly LDL metabolism and that NaNP could act either by reducing de novo synthesisrsecretion of LDL particles andror by increasing their catabolism. The hypocholesterolemic responses obtained in our study were relatively marked and comparable to the responses induced by dietary NaNP in Japanese quail fed cholesterol-enriched diet w4x. In contrast, much less pronounced increases of serum cholesterol were
E.M. Kurowska, K.K. Carrollr Biochimica et Biophysica Acta 1392 (1998) 41–50
reported earlier in experiments using NO-lowering agents w2,3x. This difference in effectiveness between dietary NO donor and inhibitors could be related to the fact that the donor was able to counteract the NO depletion caused by hypercholesterolemia. In contrast, NO-lowering agents had limited capability to act since in animals with hypercholesterolemia, blood levels of the endogenous inhibitor of NO synthase, ADMA, have been reported to be already elevated w6,7x. The hypocholesterolemia induced in rabbits by treatment with NaNP apparently differed from the acquired hypocholesterolemia observed during inflammation and tissue injury. Thus, both NaNP and inflammation caused a reduction of LDL cholesterol, but inflammation also increased plasma triacylglycerols and decreased HDL cholesterol in many species, including the rabbit w23,24x. These latter effects were likely due to release of cytokines associated with the inflammatory state w8x. Another NaNPinduced change in lipoprotein metabolism reported in our study, cholesterol ester enrichment of HDL, could be related to a specific effect of NaNP itself. Cholesterol ester ŽCE. enrichment of lipoproteins, especially VLDL and LDL, has usually been linked to accumulation of CE in the liver and was typically observed during hypercholesterolemia w25x. In contrast, dietary NaNP increased CE mainly in the HDL fraction and this occurred without altering CE and other lipids in the liver. Our in vitro studies show clearly that exposure of HepG2 cells to increasing, nontoxic concentrations of SNAP caused a dose-dependent reduction of medium level of apo B, the main protein component of LDL. This response corresponded to the LDL cholesterollowering effect of dietary NaNP, suggesting that regulation of lipoprotein metabolism by the NO-generating compounds in vivo could occur directly in the liver. The simultaneous dose-dependent increases in medium concentrations of NO end metabolites, nitrate and nitrite, indicated that in HepG2 cells, exogenous NO could be directly involved in inducing hypocholesterolemic effects. A direct role of endogenous NO in regulation of cholesterolemic responses has also been postulated in recent studies which showed that in rats treated with an NO synthase inhibitor, L-NNA, elevation of serum cholesterol was associated with reduced postprandial levels of serum
47
nitrites w3x. In our in vivo study, on the other hand, serum nitrites were not significantly increased in rabbits given NaNP, possibly because in blood samples collected from the animals 16–18 h after food withdrawal serum nitrites were already normalized. The mechanism by which SNAP suppressed overall apo B production in HepG2 cells has not been yet completely elucidated. Our study demonstrated that at the concentration of 0.5 mM, SNAP significantly decreased medium apo B but did not reduce total synthesis and secretion of cellular proteins. In agreement, incubation with SNAP at a similar concentration did not cause any reduction of total cellular protein synthesis in primary rat hepatocytes w26x. The effect of SNAP on apo B also appeared to be independent of intracellular synthesis of lipids, since SNAP at a concentration of 0.5 mM did not alter rates of 14C-acetate incorporation into cellular cholesterol, CE and triacylglycerols. This was consistent with the lack of changes in liver lipids in rabbits given NaNP, implying a similarity between mechanisms of action of NO donors in vivo and in vitro. In contrast, incubation of HepG2 cells with cytokines has been shown to increase rates of 14C-acetate incorporation into cellular sterols w8x, suggesting a difference between the hypocholesterolemic effects induced by NO donors and by inflammation. Overall, our data support the hypothesis that in HepG2 cells, SNAP suppressed net apo B production via an apo B-specific, cellular sterol-independent mechanism. However, it is still not clear whether changes in the medium level of apo B were caused by its decreased synthesisrsecretion or by increased catabolism of apo B-containing lipoproteins. Our finding that drugs that release NO can produce hypocholesterolemic responses in rabbits as well as in HepG2 cells suggested that physiological donors of NO could contribute to the maintenance of normal metabolism of lipoproteins or possibly play a role in counteracting hypercholesterolemia. However, little evidence has been found so far to support the hypothesis that exogenous L-arginine, which is a natural precursor in NO synthesis, could influence cholesterolemic responses by enhancing synthesis of NO. Our present results show that in HepG2 cells exposed to L-arginine, addition of an L-arginine analogue and NO synthase inhibitor, L-NMA, or its non-active isoform, D-NMA, to the culture medium resulted in
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equally effective reversal of apo B reduction induced by this amino acid. This suggested that both compounds counteracted the hypocholesterolemic effect of L-arginine by competing with it for transport sites across the hepatocyte plasma membrane w27x rather than by preventing its conversion to NO. In agreement with this observation, decreases in medium apo B induced by exposure of HepG2 cells to high exogenous levels of L-arginine were not associated with increased medium levels of nitratesrnitrites, confirming the absence of NO synthase activity in this cell line. The lack of involvement of NO in regulation of cholesterolemia by exogenous L-arginine has also been suggested by results in vivo. In our previous rabbit experiments, addition of L-arginine to hypercholesterolemic, amino acid-based diets reduced LDL cholesterol w28x. However, the response developed earlier than in animals given NaNP Ž after 2–3 weeks. and no associated increases were observed in plasma or liver content of NO metabolites Ž unpublished data. . In contrast, a number of other studies using proteinbased diets showed that supplementary L-arginine improved the vascular tone andror inhibited atherogenesis, largely due to its metabolism to NO w9x, but failed to reduce hypercholesterolemia w5,10,11x. This lack of cholesterolemic activity could be at least partially related to the fact that single amino acids added as supplements to protein-based diets usually have limited influence on cholesterolemia w29x. Additionally, constitutive NO synthase has been known to be poorly stimulated by physiological concentrations of L-arginine, which are much higher than its K m for this amino acid w30x. Another possibility, that a stable intermediate in NO biosynthesis from L-arginine, N–OH–Arg, could play a role in regulation of lipoprotein metabolism in the liver, merits further investigation. Previous studies documented that exogenous N–OH–Arg can enter into cultured vascular smooth muscle cells Ž which do not express a constitutive form of NO synthase. and that it can be there oxidized to NO, most likely by cytochrome P450 w14x. We therefore postulated that N–OH–Arg produced in the endothelium of liver vasculature could also diffuse into hepatocytes and there be converted into NO by a similar, constitutive NO synthase-independent mechanism. In contrast to this hypothesis, exposure of HepG2 cells to N–OH– Arg did not reduce medium levels of apo B and also
did not increase the medium content of nitratesrnitrites, suggesting that N–OH–Arg cannot be utilized as a source of NO for regulation of lipoprotein metabolism in the hepatocytes. These results should, however, be verified in vivo, since some of the metabolic responses in HepG2 cells are known to be unique for this cell line w31x, and this could include lack of enzymesrcofactors that convert N– OH–Arg into NO via the NO synthase-independent pathway.
5. Conclusion In summary, our results demonstrated that hypercholesterolemia associated with elevation of LDL cholesterol can be reduced by chronic oral administration of synthetic NO donor. However, our data did not provide evidence that a similar effect could be induced by replacing these synthetic donors with supplementary levels of a physiological NO precursor, L-arginine. The NO-generating drugs appear to affect metabolism of lipoproteins directly in the liver and most likely act by counteracting the depletion of NO caused by hypercholesterolemia, via a cellular sterol-independent mechanism. Future studies should be done to investigate in more detail how NO donors interact with metabolism of lipoproteins in the liver. These studies may lead to new therapeutic approaches aimed at reversing hypercholesterolemia and slowing the progression of cardiovascular disease.
Acknowledgements Supported by the Heart and Stroke Foundation of Ontario. The authors wish to thank Ms. Mary Moffatt, Ms. Trang Thu Tran and Ms. Debbie Friedrich for their expert technical assistance. We also extend our thanks to Dr. David Topping for his comments on the manuscript.
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