Effects of a thyromimetic on apolipoprotein B-100 in rats.

We have studied the effects of a cardiac sparing thyromimetic, CGS 23425, on postprandial levels of triglycerides, abundance of apolipoprotein B (apo B) protein and hepatic apo B mRNA expression in rats. When compared with control rats, triglyceride clearance was significantly accelerated by treatment with CGS 23425. A full return to baseline values was achieved within 8 h after ingesting a large quantity of fat, as compared to >24 h in control animals. The abundance of apo B-100 protein in CGS 23425-treated hyperlipidemic rats decreased in a dose-dependent manner, but levels of apo B-48 were not significantly affected. Like L-tri-iodothyronine (L-T(3)), treatment with 30 microg/kg CGS 23425 for 6 or 9 days decreased the levels of apo B-100 protein by 80% and 40% respectively. This change was paralleled by a 27% reduction in hepatic apo B-100 mRNA. To investigate a potential mechanism of CGS 23425 action, we measured in vitro apo B mRNA editing activity in hepatocellular extract from control or CGS 23425-treated rats. Treatment with CGS 23425 increased activity of the hepatic apo B-100 editosome, apobec-1. In human hepatoma cells which lack apobec-1 activity, apo B-100 mRNA levels remained the same in cells treated with or without the agent. In summary, these observations show that CGS 23425 decreases the levels of apo B-100 in rats. This action of CGS 23425 involves apo B-100 mRNA editing activity.

Synthesis and biological activity of phenoxyphenyl oxamic acid derivatives related to L-thyronine.

The synthesis of substituted phenoxyphenyl oxamic acid derivatives related to L-thyronine (L-T3) is described. The in vitro and in vivo cholesterol lowering and cardiovascular effects of these compounds are presented and discussed.

Beneficial effects of a novel thyromimetic on lipoprotein metabolism.

Although L-triiodothyronine (L-T3) lowers cholesterol, this hormone is not used to treat hypercholesterolemia because of its cardiotoxic effects. Thyromimetics, such as the novel compound CGS 23425, that mimic the beneficial but lack the detrimental effects of T3, may be useful in the treatment of hypercholesterolemia. To show that CGS 23425 has no cardiotoxicity, atrial contractility and force were both measured and found to be unchanged in rats treated with up to 10 mg/kg drug. The lipid lowering actions of this drug resulted in a 44% decrease in low-density lipoprotein (LDL) cholesterol in hypercholesterolemic rats treated with 10 microg/kg of the compound. Normal rats required a higher dose of 1000 microg/kg to elicit a similar 50% reduction in LDL cholesterol. Both CGS 23425 or T3 (10 nM) increased the specific binding of 125I-labeled LDL to Hep G2 cells and increased LDL receptor number by 44 and 49%, respectively. These data indicate that CGS 23425 enhances hepatic clearance of serum LDL cholesterol. Normal and fat-fed animals treated with the drug showed a dose-dependent increase in apolipoprotein AI, a protein that promotes the efflux of cholesterol from peripheral tissues. Transient transfection of a rat apolipoprotein AI promoter-chloramphenicol acetyltransferase construct, in human hepatoma cells, showed a dose-dependent increase in chloramphenicol acetyltransferase activity with EC50 values of 2 x 10(-12) M and 10(-10) M for thyroid hormone receptors beta1 and alpha1, respectively, with maximal responses at 10(-7) M. These data indicate that CGS 23425 is a thyromimetic that increases apolipoprotein AI expression via thyroid hormone receptor. In summary, CGS 23425 ameliorates hypercholesterolemia by increasing apolipoprotein A1 and the clearance of LDL cholesterol. Therefore, a compound like CGS 23425 may be useful for the prevention and reversal of atherosclerosis.

Demonstration of potent lipid-lowering activity by a thyromimetic agent devoid of cardiovascular and thermogenic effects.

A potent lipid-lowering thyromimetic (CGS 26214) devoid of cardiac and thermogenic activity was identified based on its ability to preferentially access and bind the nuclear fraction of hepatocytes over that of myocytes in culture. The difference in access achieved with CGS 26214 was at least 100-fold better for hepatocytes than for myocytes. This in vitro hepatoselectivity resulted in a compound with unprecedented in vivo lipid-lowering potency with a minimal effective dose of 1 microgram/kg in rats and dogs (approximately 25x that of L-T3). At the same time, CGS 26214 was free of any cardiovascular effects up to the highest dose tested of 25 mg/kg and 100 micrograms/kg in rats and dogs, respectively.

Synthesis and structure-activity relationships of oxamic acid and acetic acid derivatives related to L-thyronine.

Aryloxamic acids 7 and 23, (arylamino)acetic acids 29, arylpropionic acids 33, arylthioacetic acids 37, and (aryloxy)acetic acid 41 related to L-triiodothyronine (L-T3) were prepared and tested in vitro for binding to the rat liver nuclear L-T3 receptor and the rat membrane L-T3 receptor. The structure-activity relationships for these compounds are described, with 7f, 23a, 29c, 33a, 37b, and 41 showing excellent potency (IC50's of 0.19, 0.16, 1.1, 0.11, 3.5, and 0.10 nM, respectively) to the nuclear receptor and significantly lower binding affinity to the membrane receptor (IC50's > 5 microM). Some of these compounds, especially in the oxamic acid series 7 and 23, showed an unprecedented potency for methyl-substituted derivatives such as 7f and 23a. Compounds 7f and 23a showed good lipid lowering effects in rats with ED50's of 20 and 5 micrograms/kg po, respectively, and a lack of cardiac side effects in rats at doses as high as 10 and 25 mg/kg po, respectively.

The ansamycins: a novel class of hypolipidemic agents with a high affinity for lipoproteins.

The ansamycins are derivatives of 3-piperazino rifamycin with potent hypolipidemic activity in nonprimate and primate species. Since the cholesterol reduction results from increased uptake and catabolism of lipoprotein cholesterol, it was hypothesized that the hydrophobicity of the ansamycins could result in a lipoprotein association which facilitates clearance. When radiolabeled ansamycins CGP 43371 or CGS 24565 were incubated with human plasma, > 95% was lipoprotein-bound up to drug levels of 25 microM. With plasma from chow-fed rats, radiolabeled compounds again distributed with the lipoproteins. Feeding a cholesterol/cholic acid diet to rats shifted the cholesterol distribution to lower density lipoproteins and in vitro incubation resulted in a shift of radiolabeled drug to lower density lipoproteins as well. Intravenous administration of radiolabeled ansamycins to chow-fed or cholesterol-fed rats resulted in a plasma lipoprotein binding profile indistinguishable from the corresponding in vitro incubations. When [14C]-CGP 43371 bound in vitro to high density lipoprotein (HDL) was reincubated with increasing concentrations of low density lipoprotein (LDL), a concentration-dependent fall in HDL association and increase in LDL binding was observed. Thus, the ansamycins have a high affinity for all plasma lipoproteins and can transfer between lipoprotein fractions. When [125I]-labeled LDL or HDL was incubated with CGP 43371 and Hep G2 cells, the cell association of the 125I label was significantly increased in a dose-dependent manner. In addition, plasma clearance of [14C]cholesterol oleate-labeled HDL coinjected with CGP 43371 was accelerated relative to control rats and radioactivity was specifically increased in livers of CGP 43371-treated rats. The physical association of the ansamycins with lipoproteins may thus lead to subtle conformational changes and enhanced hepatic uptake.

Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL.

DiI-LDL (3,3'-dioctadecylindocarbocyanine-low density lipoprotein) has been extensively used in morphological and microscopic studies of receptor-mediated metabolism of LDL in many cell lines. To date the use of this fluorescent probe in a quantitative assay of LDL receptor activity has not been widely used in studies with multiple samples due to the lack of a practical method for quantitatively recovering cell-associated DiI. Therefore, detection by 125I-labeled LDL has remained the method of choice for assaying LDL receptor activity rapidly and reliably. In this paper, we describe a rapid, simple, and nonradioactive assay of LDL receptor activity using DiI-LDL. The increased sensitivity of this method was achieved by modifications to the labeling procedure of LDL and to the extraction of DiI from cells for subsequent fluorescence determination. These modifications did not affect the affinity of DiI-LDL toward HepG2 cells, and the assay was easily adapted to a rapid screen for LDL receptor modulators in this cell model.

Reduction of cardiovascular and thyroxine-suppressing activities of L-T3 by liver targeting with cholic acid.

This study was designed to determine whether the conjugation product of L-T3 with cholic acid would result in a liver-targeted compound (CGH 509A) with hypocholesterolemic (HC) activity significantly dissociable from cardiovascular (CV) and thyroxine-suppressing (TS) effects normally observed with thyroid hormone. Evaluation of HC activity in lipemic rats showed that CGH 509A was 6 times less potent than L-T3 with ED25 values estimated at 150 and 25 nmol/kg, respectively. CV function measured as changes in atrial rate, atrial tension and heart weight was determined in euthyroid rats. CGH 509A was at least 64 times less cardio-stimulant than L-T3 with minimum effective doses estimated at 2350 and 37 nmol/kg, respectively. TS activity was assessed in euthyroid rats as the potency of any compound to reduce plasma T4 levels. CGH 509A was 50 times less potent than-L-T3 with ED50 values estimated at 900 and 18 nmol/kg, respectively. From these results, it is clear that, while L-T3 was equally potent on HC, CV and TS activities, the HC potency of CGH 509A was at least 15 and 6 times greater than its CV and TS potencies, respectively.

Species variation in the atherogenic profile of monkeys: relationship between dietary fats, lipoproteins, and platelet aggregation.

Because lipoproteins and platelet aggregation have been implicated in atherogenesis, relative differences in the response of these variables to dietary fat saturation were compared in three species of monkeys differing in their susceptibility to atherosclerosis (cebus, rhesus, and squirrel monkeys). Both long-term (8-12 years) and short-term (8 weeks) responses to diets containing 31% fat calories were examined in the same monkeys. As expected, long-term feeding of coconut oil by comparison to corn oil produced significantly higher plasma concentrations of total cholesterol, LDL cholesterol, apoB, and triglycerides, as well as higher ratios of LDL/HDL cholesterol and apo B/apo A-I. These responses were characteristic of all species with cebus being most responsive and rhesus the least. The short-term plasma cholesterol response to animal fats (butter, lard, beef tallow) was significantly less than that to coconut oil. When fish oil was substituted for two-thirds of either corn oil or coconut oil, exceptional decreases occurred in plasma cholesterol and triglycerides, as well as in HDL cholesterol and apo A-I concentrations despite the fact that the fish oil diets contained more saturated fat and less polyenes than the corn oil diet. Platelet aggregation tended to increase with saturated fat consumption and greatly decreased with fish oil intake in all monkeys, although cebus monkeys were ten-fold more resistant to platelet aggregation than the other two species. The molecular species of platelet phosphatidylcholine (PC) varied with both the dietary fat fed and species of monkey. An inverse correlation (r = -0.60; p less than 0.001) was found between changes in one such PC molecular species (18:0-20:4) induced by diet and the platelet aggregation threshold. These results demonstrate that the lipemic and platelet responses to dietary saturated fat depend upon both the type of fat (i.e., the specific combination of dietary fatty acids, including the chain length of saturated fatty acids and the degree of polyunsaturation) and the species of monkey (genetic component) in which the response is elicited.

Lactose protects against estrogen-induced pigment gallstones in hamsters fed nutritionally adequate purified diets.

To evaluate the impact of dietary factors on gallstone induction in hamsters, male Syrian hamsters were fed for 2-8 wk purified diets that varied in type and amount of simple sugar (glucose vs. lactose, 17.5-72%), fat (2-5%), fiber (0-15%) and estrogen (0 or 300 micrograms/kg diet). Plasma and liver cholesterol and plasma triglycerides were measured, daily weight gain was determined, cecal weights were obtained, and gallbladder bile was scored by light microscopy and analyzed chemically for its lithogenicity and gallstone incidence. Lactose reduced plasma lipids, especially triglycerides, and hepatic cholesterol accumulation, and maintained a lower biliary cholesterol concentration. When fed at 30% or more, lactose reduced weight gain, increased cecal volume 2- to 4-fold and prevented gallstone formation. Diarrhea and death from 'wet tail' was associated with gallstones and was frequent in hamsters fed glucose without fiber, but its incidence was essentially eliminated by rice flour plus fiber or lactose. Under these experimental conditions of time and diet, estrogen supplementation was required for the formation of gallstones. These appeared to be pigment stones containing a minimal amount of cholesterol. In summary, pigment gallstones were induced in less than 8 wk in hamsters fed estrogen-supplemented purified diets. Lactose feeding improved lipid metabolism and reduced gallstone formation, apparently through its impact on large bowel metabolism.

Taurine modulates platelet aggregation in cats and humans.

To explore the relationship between whole-body taurine status and function, the taurine concentration in plasma and platelets was measured and evaluated in terms of ex vivo collagen-induced platelet aggregation in taurine-depleted cats and taurine-supplemented humans. Taurine status exerted a significant effect on platelet aggregability. Platelets from taurine-depleted cats were twice as sensitive to aggregation as platelets from cats receiving taurine. On the other hand, platelets from humans with normal taurine status increased resistance to aggregation by 30-70% when supplemented with taurine at 400 or 1600 mg/d, respectively. Decreased platelet aggregability was associated with increased platelet taurine and glutathione concentrations and decreased thromboxane release on aggregation. These data indicate that taurine in vivo stabilizes platelets against aggregation such that during taurine depletion platelets become overly sensitive whereas during supplementation their tendency to aggregate is depressed.

Retinal pigment epithelium possesses both LDL and scavenger receptor activity.

The lipid metabolism of photoreceptors depends in part on the retinal pigment epithelium (RPE). One aspect of cholesterol homeostasis in cultured bovine RPE was evaluated by measuring low-density lipoprotein (LDL) and scavenger receptor activity with [125I]-LDL and [125I]Ac-LDL, respectively. Incubation of RPE cells in the presence of increasing concentrations of LDL or Ac-LDL resulted in down-regulation of the LDL receptor but not the scavenger receptor, patterns consistent with the presence of both receptors on these cells. This receptor profile distinguishes the RPE cell from fibroblasts and indicates its similarity to macrophages and arterial endothelial cells.

Taurine enhances low density lipoprotein binding. Internalization and degradation by cultured Hep G2 cells.

To evaluate the impact of taurine on hepatic cholesterol catabolism low density lipoprotein (LDL) binding, internalization and degradation were measured in cultured Hep G2 cells. Preincubation of cells with 0.1-10 mM taurine for 24 h stimulated LDL receptor activity by as much as 100%. Only the high affinity LDL receptor activity (specific) was increased by taurine preincubation, whereas the low affinity receptor activity (nonspecific) remained unchanged. Scatchard analysis of the binding data revealed that taurine doubled the number of LDL receptors without affecting receptor affinity. Taurine-enhanced LDL receptor activity was most pronounced when LDL concentrations exceeded 100 micrograms/ml, but was noted at taurine concentrations as low as 0.1 mM (plasma level). Interestingly, taurine had no effect on LDL receptor activity when it was added simultaneously with 125I-LDL to Hep G2 cells, or when non-bile acid-producing human skin fibroblasts were tested. Stimulation of LDL receptor activity was also obtained with 10 mM cysteine, a taurine precursor, but not with glycine. Increased cellular concentrations of taurine and cysteine were associated with an elevated rate of bile acid synthesis and a reduced cellular free cholesterol concentration. The data suggest that taurine enhanced LDL receptor activity by sparing cysteine, a known sulfhydryl group donor and stimulator of 7 alpha-hydroxylase activity, and that the latter stimulated bile acid production leading to increased utilization of cellular free cholesterol and enhanced LDL uptake.

ApoE distribution among lipoproteins of rhesus monkeys is modulated by dietary fat and cholesterol.

The effect of dietary saturated fat and cholesterol on plasma cholesterol and apolipoprotein E (apoE) distribution among lipoproteins was studied in rhesus monkeys. Two groups of four monkeys had been fed diets containing 31% energy as either corn oil or coconut oil for 5 yr from birth. Each group was then fed short-term their respective diet with a 0.2% cholesterol supplement, the opposite fat without cholesterol, the opposite fat +0.2% cholesterol, followed by their original fat without cholesterol for 5 to 8 wk periods. Plasma was assayed for total cholesterol, total triglyclerides, and the distribution of apoE within lipoproteins (VLDL, IDL, LDL, HDL) separated by gradient-density electrophoresis. When coconut oil was fed, plasma cholesterol and triglyceride concentrations were 134% and 157%, respectively, of the levels when corn oil was fed. Cholesterol supplementation of corn oil also elevated the plasma cholesterol (141%), whereas cholesterol supplementation of coconut oil appeared to induce a synergistic increase (198%). Both groups of monkeys responded similarly to a given diet. The distribution of apoE in lipoproteins differed according to dietary treatment, with cholesterol feeding causing a major shift from HDL to IDL, whereas coconut oil caused a modest shift from HDL to VLDL. The relative amount of apoE in LDL was unchanged by diet. We conclude that dietary saturated fat or cholesterol can modulate the apoE distribution within lipoproteins in rhesus monkeys in conjunction with the previously noted expansion of the cholesteryl ester pool in VLDL and IDL.

The apo E/apo CIII molar ratio affects removal of cholesterol ester from modified human lipoproteins injected into cebus monkeys.

The removal of postprandial (PP) and postabsorptive (PA) human LDL and HDL cholesterol was examined in cebus monkeys (Cebus albifrons) following in vitro labelling of these lipoproteins by 3H-cholesterol in the presence or absence of DTNB. The removal of LDL cholesteryl ester was 3.5 and 2 times greater than that of HDL in male and female monkeys, respectively. Incubation with DTNB reduced cholesteryl ester removal by 45 and 52% for LDL and HDL, respectively. Cholesteryl ester from PA lipoproteins was removed 80% faster than that PP particles only when plasma was incubated without DTNB. Cholesterol removal from these lipoproteins was positively (r = 0.941) and significantly (P less than 0.001) correlated with the molar apo E/apo CIII ratio. The data suggest that density of lipoproteins was less important than their apoprotein composition in dictating their removal from circulation.

Evidence for distinct precursor pools for biliary cholesterol and primary bile acids in cebus and cynomolgus monkeys.

The abnormal metabolism and distribution of plasma lipoproteins have been associated with atherosclerosis and gallstones. To better understand the process of cholesterol excretion, a study was designed to determine whether the contribution of lipoprotein free 14C-cholesterol (as LDL or HDL) to biliary cholesterol or primary bile acids differs in two species of nonhuman primates, cebus and cynomolgus monkeys, having opposite plasma LDL/HDL ratios. Since amino acid conjugation might influence bile acid synthesis or secretion, the taurine and glycine conjugates of newly synthesized primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), were measured in the species capable of conjugating with taurine or glycine (cynomolgus). After total bile acid pool washout, monkeys were infused with human LDL or HDL labeled with free 14C-cholesterol, and the specific activities (SA) of biliary cholesterol and primary bile acid conjugates were determined. In both species, regardless of the lipoprotein infused, the SA of biliary cholesterol and CA were greater than those for total bile acids and CDCA, respectively. In cynomolgus, the SA of glycine conjugates was higher for CA than CDCA, while the SA of taurine conjugates was greater for CDCA than CA. Under these conditions, (i) infused lipoprotein free cholesterol (as either LDL or HDL) contributed more to biliary cholesterol than to bile acids and more to CA than to CDCA; (ii) glycine conjugated preferentially with CA rather than CDCA, while taurine was the preferred conjugate for CDCA.

Bile lipid alterations in taurine-depleted monkeys.

Newborn cebus and cynomolgus monkeys, differing in their inherent taurine-glycine conjugation of bile acids, were fed taurine-free soy protein infant formula (Isomil) with or without added taurine (500 mg/kg dry diet). After 5 months, monkeys were anesthetized, their enterohepatic circulation interrupted, and their bile pool drained for 4.5 h. Bile acid conjugation, total bile acid pool size, biliary lipid composition, and theoretical maximal cholesterol solubility in bile were determined. Taurine depletion in cebus monkey, an obligate taurine conjugator (97%), did not reduce bile acid conjugation with taurine, or did it alter bile acid pool size, biliary lipid composition, or theoretical maximal cholesterol solubility in bile. Conversely, taurine depletion in cynomolgus, a species normally conjugating with some glycine (15 to 20%), significantly reduced conjugation of taurine with bile acids from 84 to 64%, essentially doubling that of glycine from 16 to 36%. Furthermore, theoretical maximal cholesterol solubility in cynomolgus bile improved significantly as a result of taurine depletion. This improvement was associated with increased percentage distribution of biliary phospholipid from 17 to 33%, in turn reflecting an increase in the taurochenodeoxycholate to taurocholate ratio from 0.7 to 2.9. Concomitant increase in biliary cholesterol concentration associated with increased glycine conjugation precluded any changes in the percent saturation of bile which remained constant at 130% for both dietary groups of cynomolgus. Taurochenodeoxycholate uniquely conserved taurine in the face of body taurine depletion. Taurine availability thus potentially has a substantial influence on bile acid characteristics and cholesterol solubility in a glycine conjugating primate.

Growth depression in taurine-depleted infant monkeys.

In order to determine the effect of taurine depletion in primates, two species were selected which differed in their taurine conjugtion of bile acids. Consequently, eight cebus (taurine conjugators) and nine cynomolgus monkeys (glycine conjugators) were raised from birth with soybean infant milk formula lacking taurine. Half the monkeys received a 500 ppm taurine supplement. After 5 months the taurine concentration of plasma, urine and several tissues was greatly reduced in the unsupplemented monkeys. The least depletion occurred in retinal tissue of both species and in bile acids of cebus, whereas cynomolgus monkeys increased the glycine conjugation of their bile acids 125%. Taurine depletion was associated with a significant growth depression (16.8%) in the unsupplemented monkeys, but retinal degeneration was not observed. Neither species demonstrated an appreciable capacity to synthesize taurine as measured by cysteinesulfinic acid decarboxylase activity in liver and brain. The data suggest that dietary taurine is essential for maximum growth, as measured by weight gain, of infant nonhuman primates fed a soy protein milk formula.