Pii: s0955-2863(99)00008-x

Feeding the nitric oxide synthase
inhibitor L-N
nitroarginine elevates
serum very low density lipoprotein and
hepatic triglyceride synthesis in rats

Tsuyoshi Goto,* Shoko Ohnomi,* Abdelkrim Khedara,* Norihisa Kato,*
Hiroshi Ogawa,† and Teruyoshi Yanagita‡

*Department of Applied Biochemistry, Hiroshima University, Higashi-Hiroshima, Japan;†Department of Hygiene, Kinki University School of Medicine, Osaka, Japan; and ‡Department ofApplied Biological Sciences, Saga University, Saga, Japan This study was conducted to study the influence of dietary L-Nnitroarginine (L-NNA), a nitric oxide (NO)synthase inhibitor, on serum lipids and lipoproteins and on the activities of enzymes related to lipid metabolismin rats. Feeding rats a diet containing 0.2 g/kg L-NNA for 5 weeks elevated serum concentrations of triglyceride,cholesterol, phospholipid, and free fatty acid and reduced serum nitrate (an oxidation product of NO). Theelevation in serum triglyceride was mainly due to the elevation in very low density lipoprotein (VLDL)triglyceride. Contents of cholesterol and phospholipid in the VLDL fraction also were elevated by L-NNA. L-NNAtreatment caused significantly higher activity of hepatic microsomal phosphatidate phosphohydrolase (therate-limiting enzyme in triglyceride synthesis) and lower activity of hepatic carnitine palmitoyltransferase (therate-limiting enzyme in fatty acid oxidation). Activities of hepatic enzymes responsible for fatty acid synthesissuch as glucose-6-phosphate dehydrogenase, malic enzyme, and fatty acid synthase were unaffected by L-NNA.
The activity of hepatic microsomal phosphocholine cytidyltransferase (the rate-limiting enzyme in phosphatidyl-choline synthesis) was reduced significantly by L-NNA. Our results suggest that lower NO production caused theelevations in hepatic triglyceride synthesis by higher esterification of fatty acid and lower fatty acid oxidation,leading to an enrichment of VLDL triglyceride.
(J. Nutr. Biochem. 10:274 –278, 1999) Elsevier Science Inc. Keywords: nitric oxide; serum lipoproteins; hypertriglyceridemia; phosphatidate phosphohydrolase; carnitine
which in turn causes some aggravation effects such ashypertension.9 Nitric oxide (NO) is an important cellular regulator.1,2 It has Recently we have found that feeding L-N␻nitroarginine been shown to play roles in blood vessel dilation,1,2 immune (L-NNA), which is a powerful specific inhibitor of NO reactions,1,3 and the central and peripheral nervous sys- synthase, to rats caused higher concentrations of serum tems.1,2 NO production is enhanced by estrogen, inflamma- triglyceride and cholesterol and lower serum nitrate (an tion, and exercise through elevation of NO synthase activ- oxidation product of NO).10 Adding excess L-arginine to ity.4 – 8 NO is inactivated by reaction with superoxide the diet containing L-NNA elevated serum nitrate by sup- anion,1 and oxidative stress causes lower level of NO, pressing competitive inhibition of NO synthase by L-NNA,and suppressed elevations of these lipids in serum. On thebasis of these facts, we speculate that lower NO productioncauses hyperlipidemia.10 Kurowska and Carrol11 also re-ported that feeding rabbits a diet containing the NO donor Address correspondence to Dr. N. Kato, Department of Applied Biochem- sodium nitroprusside caused a reduction in low density istry, Hiroshima University, Higashi-Hiroshima, Japan.
Received September 4, 1998; accepted January 29, 1999.
lipoprotein (LDL) cholesterol and a trend of reduction in J. Nutr. Biochem. 10:274 –278, 1999 Elsevier Science Inc. 1999. All rights reserved.
655 Avenue of the Americas, New York, NY 10010 Higher VLDL by lower NO: Goto et al. serum total cholesterol. Local generation of NO within the Effect of dietary L-NNA on serum lipids and apolipoproteins epicardial coronary arteries serves to inhibit platelet adhe- sion and aggregation12 and to inhibit smooth muscle prolif- eration.13 Therefore, lower NO generation seems to lead toatherosclerosis.
Our previous study provided evidence that hypercholes- terolemia caused by L-NNA is mediated by lower synthesis of bile acid from cholesterol,14 and that hypertriglyceride- mia caused by L-NNA is due in part to lower hepatic fatty acid oxidation.10 In this study, we further examined the influence of L-NNA on serum lipoproteins and on hepatic enzymes related to triglyceride synthesis in rats.
Materials and methods
aSignificantly different from the contorl group (P Ͻ 0.05).
Male Wistar rats (Hiroshima Laboratory Animal Center, Hiro- shima, Japan) weighing 50 to 70 g were used. Animals wereindividually housed in metal cages in a temperature-controlled(24°C) room with a 12-hour light-dark cycle (lights on, 8:00 am to enzyme (ME), and fatty acid synthase (FAS) in the cytosol were 8:00 pm). All rats had free access to deionized water and experi- assayed spectrophotometrically as described by Freedland21 and mental diet. Composition of the basal diet was (in g/kg): casein, Martin et al.,22 respectively. Activity of hepatic carnitine palmi- 200; sucrose, 217; ␣-corn starch, 433; corn oil, 50; cellulose toyltransferase (CPT) in liver homogenate was measured using powder, 50; salt mixture,15 35; vitamin mixture,15 10; DL- L-carnitine, palmitoyl CoA, and 5,5Ј-dithio-bis (2-nitrobenzoic methionine, 3; and choline bitartrate, 2. L-NNA (Aldrich Chemical acid) according to the method of Bieber and Fiol.23 Company Inc., Milwaukee, WI USA) was added to the basal diet Results were expressed as means Ϯ SE and analyzed by at the level of 0.2 g/kg. After 5 weeks of consuming the diets, food was removed from the cages at 8:00 am, and the rats were lightlyanesthetized with diethylether and euthanized between 1:00 pmand 3:00 pm. Blood was collected by heart puncture, and samples were allowed to clot on ice. Serum samples were obtained bycentrifugation. Liver was immediately removed, weighed, and Gain in body weight (g/5 wk) was unaffected by L-NNA stored at Ϫ80°C until use. Portions of the fresh liver were used for feeding (P Ͼ 0.05; control 285 Ϯ 4, L-NNA 270 Ϯ 6). Food preparation of subcellular fractions.
intake (g/5 wk) also was unaffected by L-NNA (P Ͼ 0.05;control 712 Ϯ 14, L-NNA 685 Ϯ 18).
Serum concentrations of triglyceride, cholesterol, and phospholipid were higher in the L-NNA group than in the Serum lipoprotein fractions [very low density lipoprotein (VLDL), control group (P Ͻ 0.05; Table 1). Serum free fatty acid was d Ͻ 1.006 g/mL; LDL, d:1.006 –1.063 g/mL; and high densitylipoprotein (HDL), d:1.063–1.210 g/mL] were separated by step- significantly elevated in the L-NNA group, whereas serum wise density-gradient ultracentrifugation (TL-100, Beckman, San ketone bodies were unaffected by L-NNA. Serum concen- Francisco, CA USA).16 Total liver lipids were extracted by the tration of nitrate was significantly reduced by L-NNA.
method of Folch et al.17 Concentrations of triglyceride, choles- Serum concentrations of apo A-I and A-IV were signif- terol, phospholipid, and free fatty acid were measured by kits from icantly higher in the L-NNA group than the control group, Wako Pure Chemical Co. (Osaka, Japan). Concentration of ketone whereas concentrations of apo B and E were unaffected by bodies (acetoacetate and 3-hydroxybutyrate) were measured by a L-NNA. The ratio of apo B:apo A-I was unaffected by kit (Ketone Test Sanwa Chemical Institute, Nagoya, Japan).
Concentrations of serum apolipoproteins (apo A-I, A-IV, B, and E) Concentrations of triglyceride in the VLDL, LDL, and were estimated by rocket electroimmunoassay.16 To estimate NO HDL fractions were significantly higher in the L-NNA production, serum concentration of nitrate (an oxidation product ofNO) was measured by a kit (Nitrate/Nitrite Assay Kit, Cayman group than in the control group (P Ͻ 0.05; Figure 1).
Elevation in serum triglyceride by L-NNA treatment was Portions of the fresh liver from individual rats were homoge- due mainly to the elevation in VLDL triglyceride. L-NNA nized in an ice-cooled 0.25 M sucrose solution containing a 10 mM feeding also elevated VLDL cholesterol (P Ͻ 0.05), Tris-HCl buffer (pH 7.4) and 1 mM EDTA. Microsomal and whereas concentrations of cholesterol in the LDL and cytosolic fractions were prepared as described previously.18 The VLDL fractions were unaffected by L-NNA. Concentra- fractions were stored at Ϫ80°C. Protein was assayed by a kit tions of phospholipid in the VLDL and HDL fractions were (Bio-Rad Protein Assay, Bio-Rad Laboratories, Richmond, CA elevated by L-NNA (P Ͻ 0.05). Concentration of LDL USA) using bovine serum albumin as the standard.
phospholipid was unaffected by L-NNA.
Activity of Mg2ϩ-dependent phosphatidate phosphohydrolase Relative liver weight and concentrations of hepatic (PAP) in liver microsomes and cytosol was assayed as describedpreviously.18 Activities of phosphocholine cytidyltransferase cholesterol and phospholipid were unaffected by L-NNA (CTP) in the microsomes and cytosol and of choline kinase (CK) (Table 2; P Ͼ 0.05). There was a trend of elevation in liver in the cytosol were measured by the reported methods.19,20 triglyceride concentration in rats that received L-NNA Activities of glucose-6-phosphate dehydrogenase (G6PD), malic (0.05 Ͻ P Ͻ 0.1). Activities of G6PD, ME, and FAS were Distributions of triglyceride, cholesterol, and phospholipid among various lipoprotein fractions in rats fed diet with or without L-N␻nitroarginine (L-NNA). The vertical bars indicate the SE (n ϭ 10). *P Ͻ 0.05. VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL,high density lipoprotein.
unaffected by L-NNA. Activity of CPT, which is the further demonstrated that the hypertriglyceridemia by L- rate-limiting enzyme of mitochondrial ␤-oxidation, was NNA was ascribed mainly to a higher concentration of reduced significantly by L-NNA addition.
triglyceride in VLDL fraction. Concentrations of choles- The activity of microsomal Mg2ϩ-dependent PAP, which terol and phospholipid in VLDL fraction also were clearly controls the branching point in glycerolipid biosynthesis, elevated by L-NNA, but not by very much. NO appears to was elevated significantly by L-NNA, whereas the cytosolic be an important regulator of serum VLDL triglyceride.
activity was unaffected (Table 3). Microsomal activity of L-NNA treatment caused higher serum free fatty acid CTP, the rate-limiting enzyme in phosphatidylcholine bio- and lower activity of hepatic CPT (the rate-limiting enzyme synthesis, was reduced significantly by L-NNA, whereas of fatty acid oxidation) without affecting hepatic activities the cytosolic activity was unaffected. Cytosolic activity of of G6PD, ME, and FAS. This study further demonstrated CK, the first enzyme on the de novo phosphatidylcholine higher activity of PAP and lower activity of CTP in liver biosynthesis pathway, was unaffected by L-NNA.
microsomes by L-NNA. It has been suggested that PAP andCTP are involved in the rate-limiting step of triglyceridesynthesis and phosphatidylcholine synthesis, respectively, Discussion
and appear to exist in both soluble and particle forms, with Consistent with our previous study10 was the finding that the distribution of these forms being affected by the pre- L-NNA treatment caused a marked hypertriglyceridemia.
vailing metabolic status.19,24 –26 The enzymes translocate On the other hand, elevations in serum cholesterol and from cytosol to the endoplasmic reticulum to become phospholipid by L-NNA were only slight. The present study functionally active and may help to regulate glycerolipidand phospholipid metabolisms.19,25 All of these resultssuggest that dietary L-NNA causes higher triglyceride Effect of dietary L-NNA on liver lipids and the activities of enzymes relating to fatty acid synthesis and oxidation in rats Effect of dietary L-NNA on the activities of hepatic phos- phatidate phosphohydrolase, phosphocholine cytidyltransferase, andcholine kinase in rats aSignificantly different from the control group (P Ͻ 0.05).
L-NNA–L-N␻ nitroarginine.
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