Biomedical subjects
Publications and source records attributed to M Rudling.
Based on an improved molecular understanding of how bile acid metabolism is regulated, an exciting period of research developments can be expected. By new ways of stimulating cholesterol breakdown to bile acids, novel therapeutic principles can be forseen which will further improve our potential for treating and preventing atherosclerosis.
Plasma cholesterol levels increase with age, as does the incidence of coronary heart disease. The mechanisms responsible for the age-related hypercholesterolemia are not well understood. An interesting hypothesis suggests that the relative deficiency in growth hormone (GH), which occurs with aging, contributes to the development of the age-related hypercholesterolemia, because GH has beneficial effects on cholesterol metabolism. In the present work, we tested this hypothesis by the administration of GH to normal rats of varying ages. Plasma lipids and hepatic cholesterol metabolism were characterized in 2-, 12-, and 18-month-old male Sprague-Dawley rats. In 2-month-old rats, GH specifically stimulated the hepatic low density lipoprotein (LDL) receptor expression in a dose-dependent way, both at the protein level and at the mRNA level. Concomitantly, plasma cholesterol increased by approximately 30% within the large high density lipoprotein and LDL fractions. In 12-month-old animals, cholesterol 7alpha-hydroxylase (C7alphaOH) activity was reduced, whereas hepatic LDL receptors and plasma total cholesterol were unchanged. GH treatment (1 mg. kg-1. d-1) normalized the activity of C7alphaOH and had effects on plasma cholesterol and LDL receptors similar to those seen in 2-month-old animals. In 18-month-old rats, plasma cholesterol was increased 2-fold, whereas hepatic LDL receptor expression and C7alphaOH activity were similar to those of the 12-month-old animals. Infusion of GH to 18-month-old rats had similar effects on hepatic C7alphaOH and LDL receptors as seen in 12-month-old rats. However, GH treatment strongly reduced the hypercholesterolemia in 18-month-old animals. We conclude that the age-dependent increase of plasma cholesterol in rats can be reversed by the administration of GH, presumably through the pleiotropic effects of this hormone on lipoprotein metabolism.
The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7alpha-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 +/- 24 ng/mL (67% cholic acid) on day 1 to 55 +/- 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid were formed. Treatment with triidothyronine (T3) or dexamethasone alone had no significant effect on bile acid production. A combination of T3 and dexamethasone significantly increased the total bile acid production on day 4 (224 +/- 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12alpha-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7alpha-hydroxylase activity of 3.3 +/- 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T3 had an activity of 16.4 +/- 3.6 pmol/mg protein/min. The cholesterol 7alpha-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 +/- 0.9 cpm/microg total nucleic acids (tNAs). T3 or dexamethasone-treated cells did not differ from control cells, whereas the combination of T3 and dexamethasone increased the mRNA levels to 20.6 +/- 2.8 cpm/microg tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 +/- 79 ng/mL on day 2 to 1490 +/- 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7alpha-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3- to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids.
Hypercholesterolemia is a consistent feature of the nephrotic syndrome. However, the mechanisms underlying this perturbation are unclear. In the present work, we have investigated different factors that influence hepatic cholesterol metabolism using the nephrotic rat as a model. The induction of nephrosis resulted in a severe and sustained hypercholesterolemia. However, no effect on the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl CoA reductase, could be detected. Further, plasma lathosterol/cholesterol ratio, a measure of cholesterol synthesis, was not altered. Also, plasma levels of mevalonate, both a substrate for cholesterogenesis beyond the rate-limiting step and a marker for cholesterol synthesis, did not differ between control rats and those with established hypercholesterolemia. There was no detectable change in the expression of low density lipoprotein (LDL) receptor between the two experimental groups. We conclude that the early increase in cholesterol synthesis reported after the induction of nephrosis is not necessary for the maintenance of hypercholesterolemia. Established hypercholesterolemia of the nephrotic syndrome seems to represent a steady state in which neither enhanced hepatic cholesterol synthesis nor retarded LDL cholesterol clearance is of major importance.
The high-affinity degradation of low-density lipoprotein (LDL) is enhanced 3- to 100-fold in leukemic blood cells from patients with acute myelogenous leukemia (AML), suggesting an increased cellular LDL receptor expression. There are, however, inconsistencies regarding the published properties of LDL receptor regulation in AML cells, and previous data on this are indirect. In the present study the aim was to determine whether the LDL receptor number is increased in AML cells. The LDL receptor number was assayed by ligand blot with rabbit 125I-labeled beta-very-low-density lipoprotein (beta-VLDL) of transferred, SDS-polyacrylamide-gel-electrophoresis-separated AML cell membranes. Samples from 10 patients, six with AML, one with chronic myelogenous leukemia in blast crisis, and three with acute lymphoblastic leukemia, were investigated. The LDL receptor expression was strongly suppressed in all samples to levels lower than that of normal mononuclear cells. This was despite the fact that cells from one patient with AML of M4 subtype had a 50- to 100-fold higher 125I labeled LDL degradation compared with normal cells. Immunoblots with antibodies against gp330/megalin and the LDL-receptor-related protein (LRP) and ligand blot using 125I-labeled 39-kd receptor-associated protein (RAP) could not detect gp330/megalin or VLDL receptors. The LRP was abundant in AML samples of M4 and M5b subtype, as determined from both RAP ligand blot and immunoblot using an LRP-specific antibody. It is concluded that LDL receptors are suppressed in AML cells. It is possible that the high degradation of 125I-labeled LDL present in type M4 and M5 AML cells may involve another lipoprotein receptor.
Growth hormone (GH) has an important role in the regulation of hepatic LDL receptor expression and plasma lipoprotein levels. This investigation was undertaken to characterize the effects of GH on hepatic cholesterol and bile acid metabolism in the rat. In hypophysectomized (Hx) rats, the activities of the rate-limiting enzymes in cholesterol and bile acid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and cholesterol 7alpha-hydroxylase (C7alphaOH), were reduced by 71 and 64%, respectively. HMG CoA reductase mRNA levels were reduced by 37%, whereas C7alphaOH mRNA was increased by 81%. LDL receptor expression was reduced by 18% in Hx rats, without any change in the LDL receptor mRNA levels. Although the normal diurnal variation of C7alphaOH activity was preserved in Hx rats, the activity of C7alphaOH was much reduced both at midday and midnight. Total hepatic cholesterol was increased by 14% in Hx animals whereas microsomal cholesterol was unchanged. The rate of cholesterol esterification was enhanced (by 38%) in liver microsomes from Hx rats. Stepwise hormonal substitution of Hx rats showed that GH, but not thyroid hormone or cortisone, was essential to normalize the enzymatic activity of C7alphaOH. GH also normalized the altered plasma lipoprotein pattern in Hx rats, and increased the fecal output of bile acids. The latter effect was particularly evident when GH was combined with cortisone and thyroid hormone. Also in normal rats, GH stimulated C7alphaOH activity. In conclusion, GH has an essential role to maintain a normal enzymatic activity of C7alphaOH, and this, at least in part, explains the effects of GH on hepatic cholesterol metabolism. GH is also of critical importance to normalize the altered plasma lipoprotein pattern in Hx rats.
Hepatic cholesterol metabolism was studied in operative liver biopsies from 17 morbidly obese subjects and compared with that in samples from 15 nonobese controls. The aim was to understand the mechanisms causing the hypersecretion of cholesterol into bile. The content of cholesteryl esters was increased threefold in the liver of obese subjects compared with that of the controls (P < .0001). The activity and the messenger RNA (mRNA) level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme for cholesterol synthesis, were higher in the obese subjects compared with the nonobese subjects (75% and 140%, respectively; P < .01). In the obese subjects, the activity and mRNA level of cholesterol 7alpha-hydroxylase, which regulates the catabolism of cholesterol to bile acids, were also increased by 140% (P < .05) and 180% (P = .06), respectively, as compared with the controls. There was a significant correlation between the activities and the mRNA levels of cholesterol 7alpha-hydroxylase among the obese subjects (r = +0.65, P < .01). The activities of acyl-coenzyme A:cholesterol acyltransferase (ACAT), which governs cholesteryl ester formation, in obese and nonobese patients were 12.5 +/- 1.7 and 8.1 +/- 1.2 pmol/min/mg protein, respectively (P < .05), and the low-density lipoprotein (LDL) receptor mRNA levels were 5.3 +/- 0.7 and 4.5 +/- 0.9 molecules of mRNA/microg of RNA, respectively. We conclude that the activities of three key enzymes in hepatic cholesterol metabolism were increased in morbidly obese subjects compared with nonobese controls, as were mRNA levels of HMG CoA reductase and cholesterol 7alpha-hydroxylase. The mRNA level of the LDL receptor in the obese subjects was not significantly changed. The hypersecretion of cholesterol occurring in obesity is neither due to a reduced conversion of cholesterol to bile acids nor to a decreased esterification of hepatic cholesterol but may be due to an increased synthesis of cholesterol.
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Treatment with pharmacological doses of estrogen is the most potent way to stimulate hepatic LDL receptor expression in vivo. The mechanism for this effect is unclear, in part because of difficulties in inducing this stimulation in vitro. A fundamental question, whether estrogen receptors (ERs) mediate this stimulation, has not been addressed. The aim of the current study was to determine the involvement of ERs in the estrogen-induced stimulation of LDL receptors. Treatment of rats with high doses of ethynylestradiol for 7 days increased the hepatic LDL receptor protein and mRNA levels four- and threefold, respectively. LDL receptor stimulation in estrogen-treated rats was not due to their reduced food intake because hepatic LDL receptor expression did not increase in rats fasted for 72 hours. Treatment with antiestrogen (tamoxifen or clomiphene) abolished the LDL receptor stimulatory effect of ethynylestradiol at both the protein and mRNA levels. Antiestrogen alone had no effect on hepatic LDL receptor expression and did not influence the strong resistance to dietary cholesterol normally present in rats. It is concluded that ERs are critically involved in the induction of hepatic LDL receptor expression by ethynylestradiol. The known role of growth hormone for the expression of hepatic ERs may therefore play a role in the modulation of the effects of estrogen on cholesterol metabolism and hepatic LDL receptors in the rat.
We previously demonstrated that GH potentiates the biological activities of endotoxin in the rat. In the present study, we wanted to determine if the potentiating effects of GH on the biological activities of endotoxin could be reproduced by insulin-like growth factor I (IGF-I). Endotoxin (5 mg/kg BW) was injected in rats primed with or without GH or IGF-I for 3 days. As expected, endotoxin administration markedly increased circulating tumor necrosis factor (TNF) and interferon-gamma (IFN gamma) and induced organ injury, hypoglycemia, and hyperlipidemia. In GH-primed rats, endotoxin induced a further increase of serum IFN gamma (but not TNF); and five out of six of those rats died within 15 h after giving endotoxin. However, little difference between endotoxin-treated rats with and without IGF-I priming could be seen. Furthermore, IGF-I infusion altered blood glucose, urea, and circulating ICF-I levels more than GH infusion. Therefore, IGF-I does not enhance the biological activities of endotoxin in the rat, suggesting that the enhancement of endotoxin effects by GH is via an IGF-I-independent pathway. Priming rats by GH (but not by IGF-I) induced a further increased response of serum IFN gamma but not TNF to subsequent endotoxin challenge, suggesting that IFN gamma rather than TNF is likely to be involved in this process.
Histamine has been shown to be involved in atherosclerosis and coronary heart disease. Little information is available regarding the effects of histamine on lipoprotein metabolism. In the current study, we investigated the effects of histamine on the expression of hepatic low density lipoprotein (LDL) receptors and on plasma lipoproteins in the rat. Injection of compound 48/80 (C48/80, a histamine releaser) or histamine reduced hepatic LDL receptor expression, but not LDL receptor messenger RNA levels. Oral administration of polymyxin B (an antiendotoxin antibiotic and a histamine releaser) before the injection of C48/80 or histamine did not attenuate their effects. Polymyxin B itself had effects similar to those of C48/80 and histamine on LDL receptors. These results suggest that the effects of histamine are not mediated by the induction of gut-derived endotoxemia. Histamine H2 agonists (dimaprit and impromidine), but not H1 agonists (2-methylhistamine and 2-thiazolylethylamine), also reduced hepatic LDL receptor expression. The suppressive effect of C48/80 on hepatic LDL receptor expression was not attenuated by either the H1 antagonist (chlorpheniramine) or the H2 antagonist (cimetidine). Administration of C48/80 also reduced plasma high density lipoprotein (HDL) cholesterol. The H1 antagonist (chlorpheniramine), but not the H2 antagonist (cimetidine), almost completely reversed the effect of C48/80 on plasma HDL cholesterol. In conclusion, histamine suppresses hepatic LDL receptor expression via a non-H1 receptor-mediated pathway, and histamine reduces plasma HDL cholesterol via an H1 receptor-mediated pathway.
Hyperlipoproteinemia is one of the phenotypic characteristics of the fat Zucker rat that carries a mutation in the leptin receptor gene. In the present study, we studied the regulation of hepatic low density lipoprotein (LDL) receptor expression in lean and fat Zucker rats. Compared with lean rats, the fat ones had a pronounced (approximately 60%) reduction in hepatic LDL receptor expression, whereas the levels of receptor messenger RNA (mRNA) were not reduced. Fat rats had increased levels of very low density lipoproteins and high density lipoproteins, but their plasma apo B100 within LDL was reduced. Challenge with 2% dietary cholesterol for 8 days suppressed hepatic LDL receptor expression in lean animals to similar levels as seen in fat ones, whereas the reduction in mRNA levels was much less pronounced. Treatment with ethynylestradiol (5 mg/kg BW per day) for 4 days strongly stimulated hepatic LDL receptor expression in both lean and fat rats; this treatment also increased LDL receptor mRNA levels, but to a lesser extent. In conclusion, the basal expression of hepatic LDL receptors is reduced in fat Zucker rats, but the capacity for the regulation of the receptors remains intact.
Endotoxin induces hyperlipidaemia in experimental animals. In the current study, we investigated whether endotoxin alters hepatic low-density lipoprotein (LDL) receptor expression in rats. Endotoxin treatment suppressed hepatic LDL receptor expression in a dose- and time-dependent manner. Eighteen hours after intraperitoneal injection of increasing amounts of endotoxin, LDL receptor and its mRNA levels were determined by ligand blot and solution hybridization respectively. LDL receptor expression was inhibited by about 70% at a dose of 500 micrograms/100 g body weight. However, LDL receptor mRNA levels were markedly increased in all endotoxin-treated groups at this time point (by 83-136%; P < 0.001). Time-course experiments showed that LDL receptor expression was already reduced by 48% 4 h after endotoxin injection and was maximally reduced (by 63-65%) between 8 and 18 h. Changes in hepatic LDL receptor mRNA showed a different pattern. By 4 h after endotoxin injection, LDL receptor mRNA had decreased by 78% (P < 0.001). However, by 8 h after endotoxin injection, LDL receptor mRNA had returned to levels similar to controls, and 18 and 24 h after endotoxin injection, they were increased by about 60% (P < 0.05). Separation of plasma lipoproteins by FPLC demonstrated that endotoxin-induced changes in plasma triacylglycerols and cholesterol were due to accumulation of plasma apolipoprotein B-containing lipoproteins among very-low-density lipoprotein, intermediate-density lipoprotein and LDL. It is concluded that endotoxin suppresses hepatic LDL receptor expression in vivo in rats.
Growth hormone (GH) has an important role in the regulation of hepatic LDL receptor expression and plasma lipoprotein levels. This investigation was undertaken to evaluate if these effects of GH on hepatic LDL receptors are direct or mediated by insulin-like growth factor I (IGF-I). Two models were studied in which substitution with GH is important for the regulation of hepatic LDL receptors: hypophysectomized rats receiving high-dose ethynylestradiol or challenge with dietary cholesterol. The hypophysectomized rats were hormonally substituted by infusion with dexamethasone and L-thyroxine, and either GH or IGF-I. In both models, GH was essential for maintaining normal expression of LDL receptors. In contrast, despite fully normalized plasma levels, IGF-I did not support the expression of hepatic LDL receptors. Analysis of plasma lipoproteins revealed that substitution with GH, but not with IGF-I, reduced LDL and intermediate density lipoproteins. In addition, determination of hepatic mRNA levels for apo B-100 and apo B-48 indicated that GH may be more effective than IGF-I in the promotion of apo B mRNA editing. In conclusion, GH has specific effects on hepatic LDL receptor expression and plasma lipoprotein levels that are not mediated by IGF-I.
The current investigation aimed to determine if the gene expression of important structures in cholesterol metabolism is altered in a solid human tumor. For this purpose we determined low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) transcript levels and total tissue cholesterol in both tumors and normal kidney tissue in a series of samples from 29 patients with renal cell carcinoma. (RCC). LDL-receptor- and HMG-CoA reductase mRNA levels were reduced in tumor tissue as compared with normal kidney tissue (by 29%, P < 0.03, and 31%, P < 0.001 respectively). The LDL-receptor mRNA levels correlated significantly to the mRNA levels for HMG-CoA reductase in tumors (P < 0.0001) and normal kidney tissue (P < 0.03). The total tissue cholesterol levels in tumors were 5-fold elevated (P < 0.0001) as compared to normal kidney. We conclude that the mRNA levels for LDL-receptor and HMG-CoA reductase are regulated in parallel in human RCC and in normal kidney, and that these mRNA are generally reduced in human RCC. The data indicate that the accumulation of cholesterol in RCC may not be due to increased LDL receptor expression or de novo synthesis of cholesterol.
To investigate if growth hormone (GH) alters the effects of endotoxin in rats, endotoxin (5 mg kg-1 body weight) was injected into normal rats and into rats primed with GH for 3 days. Endotoxin had injurious effects on renal and liver function and induced hypoglycaemia and hyperlipidaemia as expected. GH-primed rats became extremely sick after endotoxin challenge and had more marked abnormalities of renal and liver function and much more pronounced hypoglycaemia and hyperlipidaemia than non-primed rats. It is concluded that growth hormone potentiates the in vivo biological activities of endotoxin in the rat. The results may also suggest that caution should be exerted when considering GH treatment in catabolic situations with endotoxaemia and that special attention should be paid to the prevention of infection during GH therapy in such situations.
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Bezafibrate is a hypolipidemic fibric acid derivative known to induce cholesterol supersaturation of bile. To characterize its effects on hepatic cholesterol metabolism, 31 normolipidemic, normal-weight patients with gallstones undergoing cholecystectomy were studied. Eleven patients (5 men) were randomized to treatment with bezafibrate, 200 mg three times daily for 4 weeks before operation; the remaining 20 patients (5 men) served as nontreatment controls. At operation, a liver biopsy specimen was obtained under standardized conditions and several important parameters of cholesterol metabolism were assayed. Bezafibrate treatment lowered total plasma cholesterol and triglycerides 30% and 37%, respectively. The hepatic cholesterol 7 alpha-hydroxylase activity was reduced by approximately 60% in the bezafibrate treated patients compared with the controls, whereas the acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity was similar in the two groups. The total 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity was increased twofold in the treated patients, whereas the active enzyme remained about the same as in the controls. The low-density lipoprotein (LDL) receptor binding activity was unaffected by the treatment. Bezafibrate treatment significantly reduces cholesterol 7 alpha-hydroxylase activity, and it is suggested that this may play an important role for the development of supersaturated bile during such therapy.