Spontaneous lesions in cynomolgus monkeys used in toxicity studies.

Spontaneous lesions in wild-caught, laboratory-maintained cynomolgus monkeys used in drug-toxicity studies were examined histopathologically in an effort to better distinguish toxic changes from spontaneous lesions and assess the toxicity of drugs more exactly. In the liver and kidney, where toxic changes are observed frequently, many spontaneous lesions were observed. Infiltration of mononuclear cells, vacuolization of the hepatocytes, dilatation of the renal tubules, and vacuolization of the renal epithelia were observed at a relatively high frequency. It is considered important to examine these changes carefully, because they closely resemble the changes recognized as toxic. Deposition of brownish pigment was observed in various organs such as the liver, kidney, spleen, intestinal tract, lung and brain, however the type of pigment differed among the organs, and histochemical examination revealed anthracosis or accumulation of hemosiderin, or melanin. Since the monkeys were caught in the wild, many parasitic lesions were observed especially in the large intestine and liver. Helminthous worms were frequently observed in the granulomas in the large intestine, however, no parasites were observed in the granulomas in the liver. Such lesions in the liver may be misinterpreted as toxic changes, when only scars or inflammatory lesions are observed.     

Evidence for heterogeneity of low-density lipoprotein metabolism in the cynomolgus monkey

Preliminary studies were performed to establish whether there was kinetic heterogeneity in the metabolism of subclasses of low-density lipoproteins (LDL) in the cynomolgus monkey. Previous studies of the effects of inhibition of hepatic triglyceride lipase in this species had shown an increase in the mass of lighter LDL (Sf greater than 9) and a decrease in the mass of denser LDL. LDL (1.019 less than d less than 1.063) were subdivided into two subfractions LDL1 (1.019 less than d less than 1.035) and LDL2 (1.035 less than d less than 1.063) by ultracentrifugation. The lipoproteins in these two fractions could be shown to have different flotation by analytic and isopycnic ultracentrifugation. When tracer amounts of homologous 125I-labeled very-low-density lipoproteins (VLDL) were injected into chow-fed cynomolgus monkeys, apoB radioactivity appeared in LDL1 prior to its appearance in LDL2. [125I]LDL1 injected into the monkey was removed from the LDL1 density subclass with a half-life of 5.5-10.3 h. Much of the radioactivity injected as LDL1 was converted to denser LDL (LDL2). Labeled LDL2 injected into the monkey was not converted to LDL1. Thus, at least two kinetically distinct subpopulations of LDL circulate in the plasma of this species. The lighter LDL is to a large extent a metabolic precursor of the more dense LDL (LDL2).     

Receptor-independent low-density lipoprotein catabolism. Evaluation of 2-hydroxyacetaldehyde-treated lipoprotein as a probe for its measurement

This study examines the protein modification procedures available for inhibiting receptor recognition of low-density lipoprotein (LDL). Glycosylation with glucose, idose or ribose blocks the interaction of the lipoprotein with the high-affinity LDL receptor on cultured fibroblast membranes and delays its clearance from the plasma of rabbits. However, the prolonged incubation required in the process also changes the metabolic properties of the lipoprotein. An alternative approach using 2-hydroxyacetaldehyde-treated LDL completely blocks receptor recognition. This modified tracer has the same metabolic properties as the reductively methylated lipoprotein in rabbits and appears to be a suitable probe for the measurement of the receptor-independent LDL catabolic pathway in humans.     

Receptor-independent low-density lipoprotein catabolism: Evaluation of 2-hydroxyacetaldehyde-treated lipoprotein as a probe for its measurement

This study examines the protein modification procedures available for inhibiting receptor recognition of low-density lipoprotein (LDL). Glycosylation with glucose, idose or ribose blocks the interaction of the lipoprotein with the high-affinity LDL receptor on cultured fibroblast membranes and delays its clearance from the plasma of rabbits. However, the prolonged incubation required in the process also changes the metabolic properties of the lipoprotein. An alternative approach using 2-hydroxyacetaldehyde-treated LDL completely blocks receptor recognition. This modified tracer has the same metabolic properties as the reductively methylated lipoprotein in rabbits and appears to be a suitable probe for the measurement of the receptor-independent LDL catabolic pathway in humans.     

Dietary carbohydrate type and cholesterol-induced hypercholesterolemia in cynomolgus monkeys: influence of oral antibiotic

The relationship of carbohydrate type to cholesterol-induced hypercholesterolemia and the potential role of intestinal flora in the above process were examined in 12 male cynomolgus monkeys (M. fascicularis). Semipurified diets provided two types of carbohydrates (starch or sucrose, 49% by calorie) with 0.4 mg cholesterol/kcal. Six weeks of the starch diet resulted in significantly enhanced hypercholesterolemia when compared to sucrose diet. Starch in relation to sucrose produced cholesterol enrichment of intermediate density lipoproteins and increase in low density lipoprotein particles, whereas sucrose increased high density lipoprotein constituents (phospholipids, cholesterol, and apoA-I) and triglyceride content of very low density lipoproteins. Fecal Escherichia counts were high during the starch diet as contrasted with sucrose diet, but the Escherichia, Streptococcus, and Bacteroides groups did not show differences by diet following each consecutive 4-week period of oral neomycin (107 mg/kg body wt) treatment and withdrawal. The magnitude of hypercholesterolemia during these periods also remained similar between starch and sucrose, suggesting formation of germfree metabolic characteristics. Thus, the magnitude of cholesterol-induced hypercholesterolemia can be affected by the type of carbohydrate, which may be in part determined by intestinal flora metabolism.     

Spontaneous diabetes mellitus in cynomolgus monkeys (Macaca fascicularis)

Glucose tolerance tests were performed with fourteen cynomolgus monkeys. They were divided into two groups with regard to the serum glucose level at the time of routine health-examination. Nine of them had normal glucose level (below 123 mg/dl, the normal group) and the other five monkeys exhibited hyperglycemia (the abnormal group). Fifty per cent glucose solution was administered into the saphenous vein at a dose of 4 ml/head. Blood samples were taken just before and 5, 10, 20, 30, 60 and 120 minutes after the glucose administration. K-value (K = 0.693/t 1/2 X 100) as the decreasing rate of serum glucose during from 5 to 60 minutes after the administration was calculated. Average K-value for eight monkeys of the normal group was 3.12 +/- 0.48. Both immunoreactive insulin level (IRI) and C-peptide immunoreactivity (CPR) increased just after the glucose administration and began to decrease 5 to 30 minutes after the administration in all the eight animals. Remaining one animal (No. 009) of the normal group showed 1.03 in K-value. For the abnormal group, K-value averaged 0.75 +/- 0.25. IRI was slightly higher in this group than in the 8 monkeys of the normal group. Furthermore, the abnormal group did not show any definite change of a certain trend in IRI and CPR. In conclusion, the former 8 monkeys were judged to be normal in the function of pancreatic beta-cells, and the latter 5 monkeys and No. 009 monkey were judged to be suffering from type II (noninsulin dependent) diabetes mellitus at different stages of the disease.     

Defective catabolism of low-density lipoprotein by fibroblasts from patients with I-cell disease.

Skin fibroblast cultures from patients with I-cell disease (mucolipidosis II) are characterized by multiple lysosomal enzyme deficiencies The present studies deal with the consequences of these deficiencies with respect to the metabolism of plasma low-density lipoproteins. Degradation of the protein moiety was defective in I-cells compared with control cells, but the binding and internalization of low density lipoprotein were much less affected. Measurements of low-density lipoprotein degradation in homogenates demonstrated directly for the first time a deficiency of acid proteinase activity in I-cell fibroblasts. Comparison of results in 6-h incubations with those in 24-h incubations showed accumulation of intracellular low-density lipoprotein in I-cell fibroblasts and an accelerating rate of degradation, possibly attributable to intracellular accumulation of low-density lipoprotein substrate. The significance of these findings with respect to low-density lipoprotein metabolism in vivo is discussed.     

Unaltered catabolism of desialylated low-density lipoprotein in the pig and in cultured rat hepatocytes.

Removal of the terminal sialic acid residues from many serum glycoproteins results in exposure of their penultimate galactose residues and rapid clearance from circulation by the liver. Low-density lipoprotein is a glycoprotein containing 21 galactose and 9 sialic acid residues per particle. Studies in this laboratory and others have shown that both the liver and extrahepatic tissues contribute to the degradation of low-density lipoprotein. This study was undertaken to determine whether desialylation of pig low-density lipoprotein alters its removal from circulation. Low-density lipoprotein was incubated at 37 degrees C with an agarose-bound neuraminidase, proteinase-free, from Clostridium perfringens. After 18 h at pH 5.0, 70% of the sialic acid residues were removed. The desialylated 131I-labelled and native 125I-labelled low-density lipoproteins were simultaneously injected into a pig, and their disappearance from plasma was followed for 96 h. The turnovers of the two were identical. In contrast, neuraminidase-treated fetuin was cleared about 200-fold faster than native fetuin. Studies were also performed in cultured rat hepatocytes. Rates of degradation of native and neuraminidase-treated low-density lipoprotein were similar, whereas asialo-fetuin was degraded at six to ten times the rate of native fetuin. Thus desialylation does not appear to alter low-density-lipoprotein catabolism by hepatic or extrahepatic cells.     

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.     

Study of abnormal plasma low-density lipoprotein in rhesus monkeys with diet-induced hyperlipidemia.

Male rhesus monkeys were divided into three groups: five were fed a regular primate chow diet and were used as controls; four received an "average" American diet; and five a special low-fat primate chow diet supplemented with 25% coconut oil and 2% cholesterol. In all of these animals, the plasma low-density lipoproteins (LDL) were isolated by ultracentrifugal flotation between densities of 1.019 and 1.050 g/ml. The LDL of the five control monkeys had variable molecular weights, with a mean value of 3.12 +/- 0.21 X 10(6) (range: 2.92 X 10(6) to 3.45 X 10(6)), and an average partial specific volume of 0.969 +/- 0.003 ml/g; both were assessed by flotation equilibrium analysis in the analytical ultracentrifuge. In the individual animals, however, the physical properties of LDL were invariant with time. The administration of either an "average" American diet or a coconut oil-cholesterol diet was accompanied by hypercholesterolemia associated with changes in LDL which were characterized by increases in molecular weight to 3.52 +/- 0.21 X 10(6) (average of nine monkeys) and in partial specific volume to 0.973 +/- 0.002 ml/g. These changes were particularly evident when the molecular weight of LDL from monkeys in the normolipidemic state was compared with that obtained from the same monkeys during the hyperlipidemic state. Chemical analyses revealed that the particles from the hyperlipidemic animals had a relatively higher cholesteryl ester content, a slight increase in phospholipids, and a marked decrease to nearly complete absence of triglycerides. The other lipoprotein components, protein, carbohydrate, free cholesterol, and fatty acids, did not vary significantly from those of control LDL. It is concluded that the administration of atherogenic diets causes structural changes in LDL which appear to be accounted for, at least in part, by changes in the composition of the lipid moiety. The changes in physical and chemical properties noted in the LDL of rhesus monkeys with experimentally induced hypercholesterolemia contrast with the apparent structurally normal LDL from rhesus monkeys with spontaneous hypercholesterolemia reported previously.     

Defective catabolism and abnormal composition of low-density lipoproteins from mutant pigs with hypercholesterolemia

Metabolic and chemical properties of low-density lipoproteins (LDLs) were studied in a strain of pigs carrying a specific apo-B allele associated with hypercholesterolemia and premature atherosclerosis. LDL mass was significantly greater in mutant than in control pigs (400 +/- 55 mg/dL vs 103 +/- 26 mg/dL), as was LDL cholesterol. When normal and mutant LDLs were injected into the bloodstream of normal pigs, the fractional catabolic rate (FCR) of mutant LDL was about 30% lower than that of control LDL. In mutant pigs, the mean FCRs of mutant and control LDL were similar, although they were much lower than the corresponding FCRs observed in normal pigs. The density profile of LDL particles differed in control and mutant pigs; the peak LDL flotation rate was shifted from S0f = 5.3 +/- 1.9 in controls to a more buoyant 7.4 +/- 0.5 in mutants. The elevation of LDL in the mutants was restricted to the most buoyant LDL subspecies. This subpopulation of mutant LDL was enriched with cholesteryl ester (47% vs 37%) and depleted of triglyceride, relative to LDL of similar density and size in controls. The lipid compositions of the denser LDL subpopulations (rho greater than 1.043 g/mL) were similar in mutants and controls. We conclude that the hypercholesterolemia of these mutant pigs is accounted for by defective catabolism of LDL. The buoyant cholesterol ester enriched LDL subspecies that accumulate in plasma may contribute to the accelerated atherogenesis that occurs in these animals.     

New mutations in low-density lipoprotein receptor gene in familial hypercholesterolemia patients from Petrozavodsk

Using an automated fluorescent single-strand conformation polymorphism (SSCP) analysis of the entire coding region, promoter zone, and exon-intron junctions of the low-density lipoprotein (LDL) receptor gene, we examined 80 DNA samples of patients with familial hypercholesterolemia (FH) from Petrozavodsk. We revealed mutations that might cause FH in five probands, including FH-North Karelia (c.925-931del7) mutation and four previously unknown mutations. These novel mutations included a transversion c.618T>G (p.S206R), one nucleotide insertion c.195_196insT (p.FsV66:D129X), a complex gene rearrangement c.192del10/ins8 (p.FsS65:D129X), and a single nucleotide deletion c.2191delG (p.FsV731:V736X). Three out of four novel mutations produce an open reading frame shift and the premature termination of translation. An analysis of the cDNA sequence of the LDL receptor showed that this might result in the formation of a transmembrane-domain-deficient receptor that is unable to bind and internalize the ligand. Our results suggest the absence of a strong founder effect associated with FH in the Petrozavodsk population.     

Effect of interleukin-1 alpha on lipoprotein lipids in cynomolgus monkeys: comparison to tumor necrosis factor.

Acute inflammation is associated with changes in lipoprotein metabolism. Cytokines are thought to mediate the metabolic effects of the inflammatory process. This study was undertaken to compare the effects of interleukin-1 alpha (IL-1 alpha) to tumor necrosis factor (TNF) on lipoprotein metabolism in non-human primates. Recombinant human IL-1 alpha (100 micrograms/kg), TNF alpha (20 micrograms/kg) and lipopolysaccharide (20 micrograms/kg) were injected into cynomolgus monkeys. Lipoprotein concentrations, plasma activities of post-heparin lipase (PHLA) and lecithin:cholesterol acyltransferase (LCAT) were measured prior to and 24 and 48 h after, injection. All three injections caused afebrile response in the animals. Interleukin-1 alpha had no effect on plasma lipoprotein concentrations, composition of lipoproteins or enzyme activity. In contrast, injection of TNF caused significant changes in lipoprotein concentrations. There was a 38% increase in plasma triacylglycerol and 30% decrease in plasma cholesterol at 48 h after injection. Concentrations of apolipoproteins A-I and B were decreased 20% and 44%, respectively, at 48 h. Compositional analyses of lipoprotein particles after TNF injection showed that both the LDL and HDL particles had decreased content of cholesterol ester and increased triacylglycerol after injection, and plasma activities of PHLA and LCAT were decreased. These changes were qualitatively similar to those seen after LPS injection. These data suggest that, unlike TNF, IL-1 alpha is not an important mediator of the inflammatory process on lipoprotein metabolism in cynomolgus monkeys.     

Dust mite-induced asthma in cynomolgus monkeys.

Animal models exhibiting high homology with humans at the genetic and pathophysiological levels will facilitate identification and validation of gene targets underlying asthma. In the present study, a nonhuman primate model of allergic asthma was developed by sensitizing cynomolgus monkeys to dust mite antigen. Sensitization elevated allergen-specific serum IgE and IgG levels, and peripheral blood mononuclear cells isolated from sensitized animals released IL-4, IL-5, and IL-10, but not IFN-gamma. Aerosolized allergen decreased dynamic compliance and induced airway inflammation and hyperresponsiveness to aerosolized histamine. Albuterol and dexamethasone inhibited the airway constriction and allergen-induced inflammation, respectively. Airway wall remodeling that included goblet cell hyperplasia, basement membrane thickening, and smooth muscle hypertrophy was particularly evident in neonatally sensitized animals. In contrast to animals sensitized as adults, neonatally sensitized animals exhibited increased sensitivity to adenosine and larger allergen-induced changes in airway resistance and dynamic compliance. These results demonstrate that sensitization of cynomolgus monkeys with dust mite induces asthmalike symptoms, some of which may be dependent on age at the time of sensitization.     

Role of the liver for the receptor-mediated catabolism of low-density lipoprotein in the 17 alpha-ethinylestradiol-treated rat

Effects of ethinylestradiol on LDL catabolism and plasma cholesterol were studied in the rat. Hormone treatment led to a reduction in plasma cholesterol and to an increased receptor-mediated catabolism of LDL in the liver and the adrenals. The liver was the most important organ accounting for more than 95% of the receptor-mediated tissue accumulation of LDL following estrogen treatment. It is concluded that the reduction in plasma cholesterol during treatment with ethinylestradiol is due to the stimulation of LDL receptors in the liver.     

Residualizing and non-residualizing analogues of low-density lipoprotein as iodine-123 radiopharmaceuticals for imaging LDL catabolism

Low-density lipoprotein (LDL) labeled via direct iodination or via the radioiodinated residualizing moiety tyramine-cellobiose (TC) were compared in rabbits as potential ¹²³I radiopharmaceuticals for imaging sites of LDL catabolism. The tissue deposition of ¹³¹I-TC-LDL after 24 h as determined by dissection was in the major catabolic organs (liver, adrenals, spleen), and its plasma clearance was slower in rabbits with dietary hypercholesterolemia than in normals. ¹³¹I-LDL was unsuitable as a metabolic tracer due to redistribution of catabolites and/or loss of the label before protein degradation, which resulted in little accumulation of radioactivity in catabolic organs and high thyroid uptake. The plasma clearance half-time was similar (ca 22 h) for the two compounds in normal rabbits, but was increased to about 36 h for ¹³¹I-TC-LDL and decreased to approximately 9 h for ¹³¹I-LDL in hypercholesterolemic animals. The were similar with dynamic imaging of control and hypercholesterolemic rabbits using ¹²³I-labeled analogues. ¹²³I-TC-LDL rapidly localized in the liver, with low thyroid accumulation of radioactivity. The hepatic uptake of ¹²³I-LDL was about half that of ¹²³I-TC-LDL, and thyroid sequestration of radioactivity was significant for ¹²³I-LDL but not ¹²³I-TC-LDL. These data suggest that whereas the residualizing ¹²³I-TC-LDL has a pharmacokinetic profile representative of lipoprotein metabolism, the biodistribution of the activity from injected ¹²³I-LDL is complicated by processes other than protein degradation. The results are discussed with regard to nuclear medicine applications in evaluating lipoprotein catabolism in man.     

Limited nonenzymatic glucosylation of low-density lipoprotein does not alter its catabolism in tissue culture

This study examines the effects of various degrees of chemical modification of low-density lipoprotein (LDL) on its catabolism by various cell types. Moderate glucosylation of LDL does not alter its interaction with the high-affinity receptor present on human fibroblasts at concentration of 5-2000 micrograms LDL-cholesterol/ml. Only heavily glucosylated LDL (more than 12 lysine residues glucosylated per apolipoprotein B) or LDL glucosylated in the presence of Na(CN)BH3, i.e., conditions not expected to occur in diabetes, inhibit receptor-mediated internalisation and degradation. Moderately glucosylated LDL is also readily recognized by cultured rat hepatocytes and porcine endothelial cells. Human monocyte-derived macrophages accumulate cholesteryl ester when incubated with acetylated LDL for 12 days but no enhanced cholesteryl ester formation was found when native or glucosylated LDL (3.3 lysines glucosylated per apolipoprotein B) were used.     

Aspirin induces alterations in low-density lipoprotein and decreases its catabolism by cultured human fibroblasts

Aspirin interacts in vitro with human low-density lipoprotein (LDL), which results in a decrease in free amino groups of apolipoprotein B and an increase of electrophoretic mobility of the particle. The aspirin-treated LDL was less efficiently recognized than native LDL by the apo B/E receptor of fibroblasts. These results suggest that aspirin in long-term treatment could influence the LDL-receptor pathway. However, aspirin-treated LDL did not bind to the scavenger receptor of macrophages.     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.