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Several agents can alter biliary cholesterol secretion, critical for cholesterol excretion and gallstone formation. Although salicylate effects on bile formation and gallstones have been studied, biliary lipid secretion has not been measured during oral aspirin treatment. We examined whether oral acetylsalicylic acid affects bile lipid secretion. Three groups of young rats were fed chow for 3 wk. Two of the groups then received aspirin at either 1.67 or 3.33 g/kg diet for 4 d. Serum, hepatic, and bile lipids were measured, as were enzymes of cholesterol synthesis and esterification. With oral aspirin, bile cholesterol secretion increased by 42% and hepatic cholesteryl ester content decreased by 40%. Serum cholesterol and hepatic free cholesterol did not change. To evaluate mechanisms of the cholesterol hypersecretion, hypothyroid animals fed low-fat or fish oil diets and repleted with triiodothyronine were also studied. Aspirin stimulated cholesterol secretion to a degree similar to triiodothyronine. An additive response was seen in fish oil-fed rats. Aspirin did not appear to have a primary action on 3-hydroxy-3-methylglutaryl-CoA reductase or acyl CoA:cholesterol acyltransferase activities, and had no direct effect on esterification of cholesterol by isolated hepatocytes. Aspirin may directly increase cholesterol transport into bile or have cell membrane effects which alter cholesterol transport. It remains to be determined whether the observed alterations in bile cholesterol secretion are specific to the rat or also apply to humans.

In the rat, both fish oil diet and thyroid hormone replacement are reported to augment bile cholesterol secretion out of proportion to bile flow or secretion of other bile lipids. We sought common mechanisms for these effects and evaluated the role of phospholipid fatty acid composition in the process. Methimazole-treated hypothyroid rats were fed low-fat chow or chow supplemented with 10% corn oil or fish oil, and were studied before and after thyroid hormone treatment. Serum, hepatic, and bile lipids were measured, phospholipid fatty acid composition determined, and hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity assayed. Fish oil diet stimulated cholesterol secretion into bile only after thyroid hormone was given, and this action was synergistic with that of thyroid hormone. Reduced serum cholesterol in fish oil-treated rats was associated with increased biliary cholesterol secretion and diminished hepatic cholesterol content. This suggests that augmented biliary cholesterol secretion may contribute to the fish oil-induced reduction of serum cholesterol. No definite relationship between hepatic or biliary phospholipid fatty acid composition and biliary secretion was apparent, although high bile cholesterol secretion was associated with a low percentage of hepatic and bile phospholipid linoleic acid.

Thyroid hormone lowers serum cholesterol and alters sterol metabolic processes. This laboratory has previously reported increased biliary lipid secretion as an early effect of triiodothyronine (T3) in the rat. To evaluate whether the bile lipid action of T3 is a primary or secondary effect, the isolated-perfused rat liver model was used. Red blood cells in lipid-free buffer were used to perfuse livers of euthyroid and methimazole-hypothyroid rats, as well as hypothyroid rats given T3 at intervals before perfusion. Bile flow was maintained by taurocholate perfusion. Hypothyroid rats had elevated pre-perfusion serum cholesterol compared to euthyroid (107 +/- 4 vs. 65 +/- 2 mg/dl) and decreased biliary cholesterol (0.016 +/- 0.001 vs. 0.031 +/- 0.004 mumol/g liver/h) secretion. Serum cholesterol decreased to euthyroid levels by 18 h after T3, an effect that was prevented by bile duct ligation. Bile cholesterol secretion doubled by 18 h, and reached levels twice euthyroid by 42 h, while phospholipid secretion doubled to levels just above euthyroid. The fourfold increase in biliary cholesterol secretion occurred with lipid-free perfusion and unchanging bile acid uptake or output. It occurred without a fall in hepatic lipoprotein cholesterol secretion. Blockade of cholesterol synthesis with lovastatin failed to alter T3-augmented bile cholesterol secretion. We conclude that T3 induces biliary cholesterol secretion concomitantly with the fall in serum cholesterol. This augmented biliary secretion did not appear to depend upon lipoprotein uptake, increased bile acid transport, or cholesterol synthesis. It did not occur at the expense of hepatic lipoprotein secretion. Facilitated biliary lipid secretion may be a primary effect of T3.

To further define thyroid hormone effects on bile acid synthesis and biliary lipid secretion, studies were done in chronic bile fistula rats. Euthyroid and methimazole-hypothyroid rats, with and without triiodothyronine (T3) injection, had total bile diversion for timed bile collections. With interrupted enterohepatic circulation, cholesterol absorption is negligible and bile acid secretion equals bile acid synthesis rate. Hypothyroid rats had diminished levels of bile acid synthesis and biliary secretion of cholesterol and phospholipid. Single dose T3 injection produced a 13-fold increase in bile cholesterol secretion and a 3-fold increase in phospholipid secretion, both initiated 12 h after T3. Bile acid synthesis increased by 50%, but the increase did not begin until 24 h after T3. Neither hypothyroidism nor T3 treatment abolished diurnal rhythms of bile acid synthesis and biliary lipid secretion. Inhibition of cholesterol synthesis with lovastatin resulted in a persistent 33% decrease in bile acid synthesis in euthyroid and hypothyroid rats, while bile cholesterol secretion only transiently decreased. Inhibition of cholesterol synthesis did not alter T3-induced bile cholesterol secretion, with a 10-fold increase seen. However, bile acid synthesis was not stimulated by T3 in the presence of lovastatin. We conclude that facilitated bile acid synthesis and biliary cholesterol secretion are early effects of T3 and may account for the hypocholesterolemia of T3. Cholesterol synthesis does not appear to be required for the T3-induced bile cholesterol secretion.

The drug probucol is known to reduce levels of blood cholesterol and to have antioxidant effects on lipoproteins that may alter their metabolism. While studying probucol feeding in mice, we observed that the drug lowered total hepatic, but not gut, 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase activities during the diurnal cycle. Hepatic fatty acyl:cholesterol acyl transferase activity and cholesterol content were not measurably affected by probucol. Probucol also abolished the induction of HMG CoA reductase activity that resulted from feeding of lovastatin, when activity was measured in microsomes washed free of drugs. These effects are consistent with previous reports that probucol increases fractional clearance of lipoprotein cholesterol by the liver. The findings raise the possibility that some patients with hypercholesterolemia may benefit from combined therapy with lovastatin plus probucol.

Thirty-Vella-isolated ileal segments in dogs were used to study the regulation of intestinal cholesterol synthesis. This excluded fistula enabled independent in vivo manipulation of luminal and vascular influences on mucosal cells. Segments were studied repeatedly and each animal served as its own control. Cholesterol synthesis rate was assessed by measuring mucosal activity of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Luminal cholesterol and 25-hydroxycholesterol were shown to reduce HMG CoA reductase activity to 64 +/- 7% and 42 +/- 4%, respectively, of control within 4 hr. Reductase activity in the excluded segment also responded to alterations in serum cholesterol produced by cholesterol or cholestyramine feeding. Similarly, in vitro studies showed that lipoprotein cholesterol inhibited HMG CoA reductase in mucosa from the excluded segment but not in mucosa from intact bowel. In contrast to sterols, fatty acids stimulated HMG CoA reductase activity by luminal contact. These findings suggest that the cholesterol needs of canine intestinal epithelial cells are acutely balanced by absorption and synthesis of cholesterol. Mucosal cells may also utilize lipoprotein cholesterol under certain conditions, perhaps via low density lipoprotein receptors. Fatty acid absorption stimulated cholesterol synthesis in the absence of luminal cholesterol, perhaps to facilitate chylomicron formation.

Low concentrations of thyrotropin-releasing hormone and one of its metabolites, histidyl-proline diketopiperazine, are shown to have an inhibitory effect on the activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase which occurs during organ culture of canine intestinal mucosa. Somatostatin and vasoactive intestinal polypeptide, in contrast, are shown to have no effect. Thyrotropin-releasing hormone, recently shown to be present in gut mucosa, may be a physiological regulator of intestinal cholesterol synthesis. Histidyl-proline diketopiperazine, an active metabolite of the hormone, is possibly the active agent since its inhibitory effect was observed at a concentration as low as 10(-12) M. These observations demonstrate an effect on intestinal metabolism by a neurotransmitter-like hormone at potentially physiologic concentrations.

We tested the possibility that simvastatin, a competitive inhibitor of HMG-CoA reductase related to mevinolin, might alter cholesterol saturation of gallbladder bile. Ten patients with Type IIa or IIb hypercholesterolemia underwent bile sampling before, and again after, treatment with 20 or 40 mg per day simvastatin for 7 to 13 weeks. Mean cholesterol saturation index of gallbladder bile fell from 1.01 to 0.77 during simvastatin treatment (p less than 0.01). This finding strongly suggests that treatment with HMG-CoA reductase inhibitors will not predispose to development of cholesterol gallstones. Indeed, it raises the possibility that such inhibitors might have a future role to play in treatment of gallstones.

The clear cell form of renal cell carcinoma is known to derive its histologic appearance from accumulations of glycogen and lipid. We have found that the most consistently stored lipid form is cholesteryl ester. Clear cell cancer tissue contained 8-fold more total cholesterol and 35-fold more esterified cholesterol than found in normal kidney. Cholesteryl ester appeared to be formed intracellularly since it was not membrane-bound and since oleate was the predominant form, as opposed to linoleate in lipoprotein cholesteryl esters. The cholesterol in clear cell tumors did not appear to be a result of excessive synthesis from acetate since HMG-CoA reductase (EC 1.1.1.34) activity was lower in cancer tissue than in normal kidney (2.9 +/- 0.8 vs. 7.2 +/- 1.2 pmol/mg of protein per min). In contrast, intracellular activity of fatty acyl-coenzyme A:cholesterol acyl transferase (ACAT, EC 2.3.1.26) was higher in tumor tissue than in normal kidney (2405 +/- 546 vs. 1326 +/- 301 pmol/mg of protein per 20 min) while cytosolic cholesteryl ester hydrolase activity appeared normal. Cholesteryl ester storage in clear cell renal cancer may be a result of a primary abnormality in ACAT activity or it may be a result of reduced release of free cholesterol (relative to cell content) with a secondary elevation in ACAT activity.

Clear cell renal cancer contains a large quantity of cholesterol ester (300-mg./gm. protein). To determine whether abnormalities in cholesterol transport could account for this sterol accumulation, the uptake, release, and imaging capabilities of intravenously injected 131I-6-iodomethyl-29-norcholesterol, a cholesterol analogue, were studied preoperatively in five patients with clear cell renal cancer. At surgery, samples of the liver, tumor, adrenal, and non-tumor kidney were obtained for analysis. 131I-sterol uptake by the tumor, when normalized for cholesterol content, was less than for adrenal, liver or kidney. In contrast, release of preloaded 131I-sterol from the human tumors was consistently slower than for normal kidney. The reduced release of free cholesterol from renal cancer cells may, in part, be responsible for the accumulation of cholesterol in human renal cancer.

In an effort to define the mechanism by which thyroid hormone increases the synthesis of hepatic cholesterol, we have investigated both in hypophysectomized and methimazole-treated hypothyroid rats the time course of T3 effects on plasma cholesterol concentration, total hepatic cholesterol, the rate of biliary secretion of cholesterol, bile acids, and phospholipids, and the activity and mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, the rate-limiting enzyme in the hepatic synthesis of cholesterol. A single dose of 200 micrograms T3 was estimated to maintain at least 90% nuclear occupancy for the ensuing 54 h of the experiment. In both preparations the relative rise in biliary secretion of cholesterol exceeded that of other biliary constituents and preceded by 12 h an increase in HMG-CoA reductase enzyme activity and its mRNA. The level of total hepatic cholesterol remained constant throughout the experiment. We interpret these findings to suggest that T3-stimulated cholesterol synthesis is mediated by an antecedent T3-induced rise in biliary cholesterol secretion. We postulate that biliary cholesterol secretion is augmented by an intrahepatic shift of cholesterol and depletion of the hepatic sampling center responsible for the feedback regulation of cholesterol synthesis. The level of HMG CoA reductase mRNA appeared to govern enzyme activity in both preparations, but the ratio of mRNA to hepatic enzyme activity was substantially greater in the methimazole-treated compared with the hyphophysectomized animals.

The regulation of intestinal cholesterol synthesis was studied utilizing canine ileal mucosa maintained in organ culture for 6 h. Viability was monitored by light and electron microscopy, measurement of cellular enzymes, and the ability to actively transport a glucose analogue. The activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.4.3.4), the rate-limiting enzyme of cholesterol synthesis, increased 4-fold during a 6-h culture. A parallel increase occurred in the rate of acetate incorporation into digitonin-precipitable sterols during this period. This increase could be prevented by the addition of cycloheximide to the culture. Pure cholesterol, 7-ketocholesterol, and 25-hydroxycholesterol, when present during the last 4 h of culture, also caused significant suppression of the rise in HMG-CoA reductase activity (final HMG-CoA reductase with the three sterols was 77 +/- 4%, 68 +/- 5%, and 58 +/- 3% of control postculture value). Bile salts at low, nontoxic concentrations also inhibited the increase of enzyme activity (2 mM taurocholate = 63 +/- 3% of control, 0.5 mM taurochenodeoxycholate = 76 +/- 6% of control). In contrast, dog lipoproteins separated by ultracentrifugation failed to significantly affect intestinal cholesterol synthesis in these short term organ cultures.