Ilona Staprans

Infection and Inflammation Induce LDL Oxidation In Vivo

Epidemiological studies have shown an increased incidence of coronary artery disease in patients with chronic infections and inflammatory disorders. Because oxidative modification of lipoproteins plays a major role in atherosclerosis, the present study was designed to test the hypothesis that the host response to infection and inflammation induces lipoprotein oxidation in vivo. Lipoprotein oxidation was measured in 3 distinct models of infection and inflammation. Syrian hamsters were injected with bacterial lipopolysaccharide (LPS), zymosan, or turpentine to mimic acute infection, acute systemic inflammation, and acute localized inflammation, respectively. Levels of oxidized fatty acids in serum and lipoprotein fractions were measured by determining levels of conjugated dienes, thiobarbituric acid-reactive substances, and lipid hydroperoxides. Our results demonstrate a significant increase in conjugated dienes and thiobarbituric acid-reactive substances in serum in all 3 models. Moreover, LPS and zymosan produced a 4-fold to 6-fold increase in conjugated diene and lipid hydroperoxide levels in LDL fraction. LPS also produced a 17-fold increase in LDL content of lysophosphatidylcholine that is formed during the oxidative modification of LDL. Finally, LDL isolated from animals treated with LPS was significantly more susceptible to ex vivo oxidation with copper than LDL isolated from saline-treated animals, and a 3-fold decrease occurred in the lag phase of oxidation. These results demonstrate that the host response to infection and inflammation increases oxidized lipids in serum and induces LDL oxidation in vivo. Increased LDL oxidation during infection and inflammation may promote atherogenesis and could be a mechanism for increased incidence of coronary artery disease in patients with chronic infections and inflammatory disorders.

Oxidized Lipids in the Diet Accelerate the Development of Fatty Streaks in Cholesterol-Fed Rabbits

Studies have indicated that oxidized lipoproteins may play a role in atherosclerosis. We have recently demonstrated that the levels of oxidized lipoproteins in the circulation can be directly correlated to the quantity of oxidized lipids in the diet. The present study tested the hypothesis that dietary oxidized lipids accelerate the development of atherosclerosis. For 12 to 14 weeks, 36 male New Zealand White rabbits were fed a low-cholesterol (0.25%) diet containing either 5% unoxidized corn oil (control diet) or 5% oxidized corn oil (oxidized-lipid diet). Serum cholesterol levels increased to a similar extent in both groups, with the majority of the cholesterol in the beta-migrating very low density lipoprotein (beta-VLDL) fraction. Beta-VLDL from control animals contained 3.86+/- 0.57 versus 9.07 +/- 2.14 nmol conjugated dienes per micromol cholesterol (P<.05) in rabbits fed the oxidized-lipid diet. No difference in oxidized lipid levels was detected in LDL. Most important, feeding a diet rich in oxidized-lipid resulted in a 100% increase in fatty streak lesions in the aorta. Additionally, rabbits that were fed the oxidized-lipid++ diet had a >100% increase in total cholesterol in the pulmonary artery that was primarily due to an increase in cholesteryl ester. Oxidized lipids are frequently present in the typical US diet, and our results suggest that consumption of these foods may be an important risk factor for atherosclerosis.

Proteinuria, not altered albumin metabolism, affects hyperlipidemia in the nephrotic rat.

It has been established previously that nephrotic hyperlipidemia is characterized by both an increase in lipid synthesis and a defect in removal of lipoproteins. The relationship between these defects and altered albumin metabolism is uncertain. One hypothesis is that hepatic lipogenesis increases in parallel with albumin synthesis. To test this hypothesis, albumin synthesis was increased in nephrotic rats fed an 8.5% protein diet (LPN) by increasing dietary protein to 40% (HPN). Proteinuria was modulated in half of the rats fed 40% protein by enalapril (HPE). Albumin synthesis was the same in both HPN and HPE, but proteinuria was reduced in HPE compared to HPN, and so were serum cholesterol and triglycerides (TG). To examine the effect of serum albumin on lipid clearance in the absence of proteinuria, plasma clearance of chylomicrons (CM) and VLDL was measured in Nagase analbuminemic rats (NAR) and found to be no different than in normal SD rats. When proteinuria was induced in NAR and in SD rats, a severe and identical defect in both CM and VLDL clearance was acquired in both groups and blood lipid levels were increased to a similar degree in both groups. Neither hyperlipidemia nor defective removal of lipoproteins from the circulation are linked to albumin synthesis or serum albumin concentration but result, at least in part, from proteinuria. Postheparin lipoprotein lipase (LPL) activity was reduced slightly in nephrotic animals compared to nonnephrotic controls, but the most striking finding was a highly significant decrease in postheraprin LPL activity in normal NAR compared to SD rats (P less than 0.001), suggesting that reduced LPL activity is not responsible for reduced clearance of CM and VLDL in nephrotic rats.

Oxidized Cholesterol in the Diet Accelerates the Development of Aortic Atherosclerosis in Cholesterol-Fed Rabbits

Oxidized lipoproteins may play a role in atherosclerosis. Recently, we have demonstrated that the levels of oxidized fatty acids in the circulation correlate directly with the quantity of oxidized fatty acids in the diet and that dietary oxidized fatty acids accelerate atherosclerosis in rabbits. The present study tests the hypothesis that oxidized cholesterol in the diet accelerates the development of atherosclerosis. Rabbits were fed a diet containing 0.33% nonoxidized cholesterol (control diet) or the same diet containing 0.33% cholesterol of which 5% was oxidized (oxidized diet). Serum cholesterol levels increased to a similar extent in both groups, with the majority of cholesterol in the beta-VLDL fraction. Moreover, in the serum beta-VLDL fraction and liver, there was a significant increase in the oxidized cholesterol levels. Most importantly, feeding a diet enriched in oxidized cholesterol resulted in a 100% increase in fatty streak lesions in the aorta. Western diets contain high concentrations of oxidized cholesterol products, and our results suggest that these foods may be a risk factor for atherosclerosis.

Effect of tumor necrosis factor (TNF) on lipid metabolism in the diabetic rat. Evidence that inhibition of adipose tissue lipoprotein lipase activity is not required for TNF-induced hyperlipidemia.

Tumor necrosis factor (TNF) administration produces an increase in plasma triglycerides that may be due to inhibition of adipose lipoprotein lipase activity and/or a stimulation of hepatic lipogenesis. We now report that TNF administration to insulinopenic diabetic rats increases serum triglycerides (2 h, 2.4-fold; 17 h, 4.3-fold). Adipose tissue lipoprotein lipase activity was markedly decreased in diabetic animals compared with controls and was not further inhibited by TNF. Incorporation of tritiated water into fatty acids in the liver was increased 45% 1-2 h after TNF and 87% at 16-17 h. These results indicate that the TNF-induced increase in circulating lipid levels can occur in the absence of a TNF-induced inhibition of adipose tissue lipoprotein lipase activity. Moreover, the clearance from the circulation of triglycerides in chylomicrons was similar in control and TNF-treated animals; these results provide further evidence that the removal of triglyceride-rich lipoproteins is not altered in the TNF-treated animals. Our data suggest that the TNF-induced stimulation of hepatic lipid synthesis may play an important role in the increase in serum triglycerides. In addition, TNF administration to diabetic animals leads to an elevation in serum glucose levels (73% at 17 h) without a change in serum insulin levels. Thus, TNF stimulation of hepatic lipogenesis is independent of changes in insulin.

Effect of interleukin-1 on lipid metabolism in the rat. Similarities to and differences from tumor necrosis factor.

Infection and inflammation are associated with hypertriglyceridemia, which is thought to be mediated by cytokines. Previous studies at our laboratory and others have shown that tumor necrosis factor acutely increases serum triglyceride levels primarily by stimulating hepatic lipid synthesis and secretion. The role of interleukin-1 (IL-1), a cytokine that is also secreted by stimulated macrophages and that has many actions that overlap those of tumor necrosis factor, has not been studied in depth. The present study demonstrates that IL-1, at doses similar to those that cause fever and anorexia and that stimulate adrenocorticotropic hormone secretion, rapidly increases serum triglyceride levels; this elevation persists for at least 17 hours. Serum cholesterol levels are not altered by IL-1. Neither is the clearance of triglyceride-rich lipoproteins affected by IL-1. However, hepatic triglyceride secretion, measured by the Triton WR-1339 technique, is increased in IL-1-treated animals. Accompanying this stimulation in hepatic lipid secretion is an increase in de novo fatty acid synthesis in the liver. IL-1 does not increase serum free fatty acid and glycerol levels, suggesting that IL-1 does not stimulate lipolysis in vivo. Additionally, inhibition of lipolysis does not prevent the increase in serum triglyceride levels, providing further evidence that lipolysis does not play a crucial role in the increased hepatic lipid synthesis and secretion induced by IL-1. In contrast, tumor necrosis factor increases lipolysis, which contributes to the increase in serum triglycerides. That multiple cytokines rapidly elevate plasma triglyceride levels suggest that these changes in lipid metabolism may play an important role in the organisms response to infection and inflammation.

Oxidized Cholesterol in the Diet Accelerates the Development of Atherosclerosis in LDL Receptor– and Apolipoprotein E–Deficient Mice

The aim of the current study was to determine whether oxidized cholesterol in the diet accelerates atherosclerosis in low density lipoprotein receptor- (LDLR) and apolipoprotein E- (apo E) deficient mice. Mice were fed either a control diet or a diet containing oxidized cholesterol. For LDLR-deficient mice, the control diet consisted of regular mouse chow to which 1.0% cholesterol was added. The oxidized diet was identical to the control diet except that 5% of the added cholesterol was oxidized. In apo E-deficient mice, the control diet contained 0.15% cholesterol, whereas in the oxidized diet, 5% of the added cholesterol was oxidized. LDLR-deficient and apo E-deficient mice were fed the experimental diets for 7 and 4 months, respectively. In mice fed the oxidized-cholesterol diets, the levels of oxidized cholesterol in sera were increased. At the end of the experiment, aortas were removed and atherosclerosis was assessed. We found that in LDLR-deficient mice, feeding of an oxidized-cholesterol diet resulted in a 32% increase in fatty streak lesions (15.93+/-1.59% versus 21.00+/-1.38%, P<0.03). Similarly, in apo E-deficient mice, feeding of an oxidized-cholesterol diet increased fatty streak lesions by 38% (15.01+/-0.92% versus 20. 70+/-0.86%, P<0.001). The results of the current study thus demonstrate that oxidized cholesterol in the diet accelerates fatty streak lesion formation in both LDLR- and apo E-deficient mice.

The effect of oxidized lipids in the diet on serum lipoprotein peroxides in control and diabetic rats.

The levels of oxidized serum lipoproteins are increased in humans and animals with diabetes. We have examined the contribution of dietary oxidized lipids on the levels of oxidized lipoproteins. In both control and streptozocin induced diabetic rats, the oxidized lipid content of mesenteric lymph chylomicrons (CM) increased when increasing quantities of oxidized lipids were administered intragastrically. However, at all levels of administered oxidized lipids, the quantity of oxidized lipids in CM was greater in the diabetic animals. These results indicate that oxidized lipids are absorbed and packaged into CM and suggest that there is increased absorption of oxidized lipids in diabetic animals. In nondiabetic rats fed a fat-free diet, the levels of oxidized lipids in their serum lipoproteins were very low. When oxidized lipids were added to the diet, the quantity of peroxides in serum lipoproteins increased about fivefold. In diabetic animals fed a fat-free diet, there were also very low levels of oxidized lipids in their serum lipoproteins, and there was no difference between control and diabetic rats. However, when diabetic animals were fed a diet containing oxidized lipids, the quantity of oxidized lipids in their serum lipoproteins increased 16-fold and were significantly greater than in controls. Thus, in both control and diabetic rats the quantity of oxidized lipids in the diet largely determines the levels of oxidized lipids in circulating lipoproteins. However, in diabetic animals the effect of diet is more pronounced. Together with the CM studies, these results demonstrate that dietary oxidized lipids make a major contribution to the levels of oxidized lipids in circulating lipoproteins and indicate that increased absorption of oxidized lipids in diabetic animals may play a role in the elevation of oxidized lipoproteins observed in this disorder.

Apoprotein composition of plasma lipoproteins in uremic patients on hemodialysis.

Abstract Soluble apolipoproteins were isolated from very low density and high density lipoproteins obtained from the plasma of uremic patients on hemodialysis. Quantitative gel electrophoresis was utilized to determine the relative amounts of lipoprotein lipase activator (ApoC-II) and inhibitor (ApoC-III) peptides of very low density lipoproteins from anephric patients, patients with glomerulo-nephritis, polycystic disease and appropriate normal subjects. The relative amounts of A and C apoproteins of high density lipoproteins from anephric patients, patients with glomerulonephritis and normal subjects were also determined. The total protein in very low density lipoproteins was increased by 300–450% and the ApoC-III/ApoC-II ratio increased from 3.4 in normal subjects to 6.2–8.4 in patient groups. The total protein in high density lipoproteins was decreased approximately 30% for all patient groups. There was no difference in the relative percentage of apoproteins A-I and A-II in the high density lipoproteins between the normal and the patient groups. However, the ratio of ApoC-III/ApoC-II was elevated in patients. Our results suggest the possibility that alterations in the composition of the C group of apoproteins of very low density lipoproteins and high density lipoproteins may contribute to the increase in triglyceride concentration by modulating the activity of lipoprotein lipase. Since anephric patients showed patterns identical to other kidney patients, it appears that the uremic state and not a specific kidney disease is associated with the apoprotein alterations.

Tumor Necrosis Factor–Increased Hepatic Very-Low-Density Lipoprotein Production and Increased Serum Triglyceride Levels in Diabetic Rats

Previous studies demonstrated that administration of tumor necrosis factor (TNF) to diabetic rats rapidly increases serum triglyceride levels and stimulates hepatic lipogenesis without affecting the activity of adipose tissue lipoprotein lipase or serum insulin levels. The purpose of this study was to determine the mechanism by which TNF increases serum triglyceride levels and stimulates hepatic fatty acid synthesis in diabetic animals. The maximal increase (∼2-fold) in serum triglyceride levels in diabetic rats is seen with a dose of 10 μg TNF/200 g body wt, and the halfmaximal effect is observed with 5 μg TNF/200 g body wt. The clearance of labeled triglyceride-rich lipoproteins from the circulation is not affected by TNF administration (triglyceride t½: diabetic vs. TNFadministered diabetic, 3.5 ± 0.7 vs. 4.0 ± 0.6 min, respectively; NS). The production of triglyceride, measured by the Triton WR-1339 technique, is increased twofold in diabetic animals after TNF administration. These results indicate that the rapid increase in serum triglyceride levels after TNF treatment is accounted for by increased hepatic lipoprotein secretion. TNF administration did not alter either the amount or activation state of hepatic acetyl-CoA carboxylase, a key regulatory enzyme in fatty acid synthesis. There was also no change in the hepatic levels of fatty acyl-CoA, an allosteric inhibitor of acetyl-CoA carboxylase. However, there was a 71% increase in hepatic citrate concentrations. Citrate is an allosteric activator of acetyl-CoA carboxylase, and changes in hepatic citrate concentrations have been shown to mediate changes in the rates of fatty acid synthesis. These results suggest that the TNF-induced stimulation of hepatic lipogenesis is mediated by citrate activation of acetyl-CoA carboxylase. At 2 h after TNF administration, neither serum glucose nor p-hydroxybutyrate levels were adversely altered in the TNF group, indicating that the disturbances in lipid metabolism are not dependent on alterations in glycemic control. The increases in serum triglyceride levels that occur during infections or stress in diabetes may be secondary to TNF.