Muricholic bile acids are potent regulators of bile acid synthesis via a positive feedback mechanism.

OBJECTIVE: Bile acid (BA) synthesis is regulated by negative feedback end-product inhibition, initiated by farnesoid X receptors (FXRs) in liver and gut. Studies on cholic acid (CA)-free Cyp8b1(-/-) mice have concluded that CA is a potent suppressor of BA synthesis. Cyp8b1(-/-) mice have increased BA synthesis and an enlarged BA pool, a phenotype shared with bile-duct-ligated, antibiotics-administered and with germ-free mice. Studies on such mice have concluded BA synthesis is induced due to reduced hormonal signalling by fibroblast growth factor (FGF)15 from intestine to liver. A mutual finding in these models is that potent FXR-agonistic BAs are reduced. We hypothesized that the absence of the potent FXR agonist deoxycholic acid (DCA) may be important for the induction of BA synthesis in these situations. DESIGN: Two of these models were investigated, antibiotic treatment and Cyp8b1(-/-) mice and their combination. Secondary BA formation was inhibited by ampicillin (AMP) given to wild-type and Cyp8b1(-/-) mice. We then administered CA, chenodeoxycholic acid (CDCA) or DCA to AMP-treated Cyp8b1(-/-) mice. RESULTS: Our data show that the phenotype of AMP-treated wild-type mice resembles that of Cyp8b1(-/-) mice with fourfold induced Cyp7a1 expression, increased intestinal apical sodium-dependent BA transporter expression and increased hepatic BA levels. We also show that reductions in the FXR-agonistic BAs CDCA, CA, DCA or lithocholic acid cannot explain this phenotype; instead, it is likely due to increases in levels of α- and ß-muricholic BAs and ursodeoxycholic acid, three FXR-antagonistic BAs. CONCLUSIONS: Our findings reveal a potent positive feedback mechanism for regulation of BA synthesis in mice that appears to be sufficient without endocrine effects of FGF15 on Cyp7a1. This mechanism will be fundamental in understanding BA metabolism in both mice and humans.

LXRß activation increases intestinal cholesterol absorption, leading to an atherogenic lipoprotein profile.

OBJECTIVES: Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRß, with overlapping but not identical functions, we investigated whether LXRα and LXRß exert different effects on intestinal cholesterol absorption. DESIGN: Wild-type (WT), LXRα(-/-) and LXRß(-/-) mice were fed control diet, 0.2% cholesterol-enriched diet or 0.2% cholesterol-enriched diet plus the LXR agonist GW3965. RESULTS: When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα(-/-) mice, but not in the WT or LXRß(-/-) animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα(-/-) mice. Greater increases in the apolipoprotein (apo)B-containing lipoproteins in serum were seen in the LXRα(-/-) mice. A 0.2% cholesterol +GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree. CONCLUSIONS: In the intestine, LXRα and LXRß seem to exert similar effects on expression of cholesterol-transporting proteins such as Npc1l1. Selective activation of LXRß may generate effects such as increased cholesterol absorption and elevated serum levels of apoB-containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRß-specific pathway might exist in terms of cholesterol transportation in addition to the main pathway.

HIV-infected subjects with the E4 allele for APOE have excess dementia and peripheral neuropathy.

HIV produces a chronic viral infection of the central nervous system that elicits chronic glial activation and overexpression of glial cytokines that are also implicated in Alzheimer disease (AD) pathogenesis. A genetic risk factor for AD is the E4 isoform for apolipoprotein E (APOE). Here we compare the frequency of neurologic symptoms for subjects with and without the E4 isoform (E4(+)and E4(-), respectively) in an HIV cohort. Compared with E4(-) subjects, twice as many E4(+) subjects were demented (30% compared with 15%) or had peripheral neuropathy (70% compared with 39%) at least once, and they had threefold more symptomatic examinations (13% compared with 3% and 42% compared with 14%, respectively)(P < 0.0001). Thus, neurologic symptoms for HIV-infection and AD are linked through an etiologic risk factor. Long-term survivors of HIV infection with E4 may be at high risk for AD; conversely, gene-viral interactions may speed AD pathogenesis.

Regulation of rat hepatic low density lipoprotein receptors. In vivo stimulation by growth hormone is not mediated by insulin-like growth factor I.

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.

On the mechanism of stimulation of cholesterol 7 alpha-hydroxylase by dietary cholesterol.

In agreement with previous work, treatment of rats with cholesterol, 2% in diet, stimulated the cholesterol 7 alpha-hydroxylase activity more than 2-fold. With less than 1% in diet, no significant effect was obtained. Intravenous infusion of cholesterol-enriched Intralipid had no stimulatory effect. In accordance with some recent work by other groups, it was shown that the stimulation of the cholesterol 7 alpha-hydroxylase by dietary cholesterol was associated with elevated levels of mRNA corresponding to the enzyme. Most of the stimulation of the activity induced by dietary cholesterol could not be prevented by lymphatic drainage. Feeding lymph fistulated rats with 2% cholesterol in diet stimulated the cholesterol 7 alpha-hydroxylase almost 2-fold, indicating that under the conditions employed, a major part of the cholesterol-induced stimulation of the activity was due to factor(s) unrelated to the flux of cholesterol from the intestine to the liver. There was a good correlation between the amount of cholesterol excreted in faeces and the activity of the cholesterol 7 alpha-hydroxylase. The half-life of intraperitoneally administered labelled cholic acid was significantly shorter in rats treated with 2% cholesterol in diet (t1/2 = 1.2 +/- 0.1 days) than in control rats (t1/2 = 1.9 +/- 0.18 days). A notable finding was that the weight of faeces was considerably higher in rats fed cholesterol than in the controls. It is hypothesized that a high dietary load of cholesterol causes increased binding of bile acids in the intestine and increased loss of bile acids in faeces. This leads to a reduced suppression of the cholesterol 7 alpha-hydroxylase by the bile acids. The results support the contention that the flux of bile acids rather than the flux of cholesterol from the intestine is the major direct regulator of bile acid biosynthesis.

Rabbit liver contains one major sterol 12alpha-hydroxylase with broad substrate specificity.

Conversion of cholesterol into cholic acid in mammalian liver requires a 12alpha-hydroxylation step. Results have been presented suggesting that two different enzymes are involved in this hydroxylation with different activities towards the two steroids believed to be the physiological substrates for the enzyme, 7alpha-hydroxy-4-cholesten-3-one and 5beta-cholestane-3alpha,7alpha-diol. It is shown here that rabbit liver microsomes and partly purified sterol 12alpha-hydroxylase as well as COS cells transfected with a cDNA coding for this enzyme are able to catalyze 12alpha-hydroxylation of the two substrates at similar relative rates. Also 7alpha-hydroxycholesterol and 3alpha,7alpha-dihydroxy-5beta-cholestanoic acid are 12alpha-hydroxylated by the three systems. It is concluded that rabbit liver contains one major sterol 12alpha-hydroxylase with a broad substrate specificity.

Clofibrate treatment increases stearoyl-CoA desaturase mRNA level and enzyme activity in mouse liver.

Stearoyl-CoA desaturase activity is encoded by two highly homologous genes, SCD1 and SCD2, which show tissue-specific expression and regulation. SCD1, which is expressed in the liver, showed a marked diurnal variation with the highest expression during the feeding period. Treatment of mice with the peroxisome proliferator clofibrate, which induces several lipid metabolizing enzymes, increased both the enzyme activity and mRNA level in the liver, indicating regulation at the transcriptional level. The highest expression of both SCD1 and SCD2 was found in brown adipose tissue, which was slightly down-regulated by feeding a fat-free diet.

Thyroid hormone suppresses hepatic sterol 12alpha-hydroxylase (CYP8B1) activity and messenger ribonucleic acid in rat liver: failure to define known thyroid hormone response elements in the gene.

Sterol 12alpha-hydroxylase (CYP 8B1) is a microsomal cytochrome P450 enzyme involved in bile acid synthesis that is of critical importance for the composition of bile acids formed in the liver. Thyroidectomy of rats caused a more than twofold increase of CYP8B1 and an almost fourfold increase of the corresponding mRNA levels compared to sham-operated rats. Treatment of intact rats with thyroxine caused a 60% reduction of enzyme activity and a 50% reduction of mRNA levels compared to rats injected with saline only. To investigate whether the promoter of the gene contains thyroid hormone response elements, the complete structure of the rat gene was defined. In similarity with the corresponding gene in mouse, rabbit and man, the rat gene was found to lack introns. It had an open reading frame containing 1500 bp corresponding to a protein of 499 amino acid residues. Although thyroid hormone decreased CYP8B1 activity and mRNA in vivo, no hitherto described thyroid hormone response elements were identified 1883 bases upstream of the transcription start site. It is concluded that rat CYP8B1 is regulated by thyroid hormone at the mRNA level. The results are discussed in relation to the structure of the gene coding for the enzyme.

Genetic analysis of a Japanese cerebrotendinous xanthomatosis family: identification of a novel mutation in the adrenodoxin binding region of the CYP 27 gene.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive lipid-storage hereditary disorder, is caused by mutations in the sterol 27-hydroxylase gene (CYP 27). A 24-year-old female Japanese CTX patient and her parents were studied for a CYP 27 mutation. Multiple xanthomas were the main complaint of the patient and plasma cholestanol level was markedly elevated. Sterol analysis of a xanthoma biopsy confirmed cholesterol and cholestanol deposition, and the cholestanol accounted for 8.1% of the total sterols. Sterol 27-hydroxylase activity in fibroblasts derived from the patient was undetectable, while the activities in fibroblasts from her mother and father were 54% and 41% of the normal level, respectively. Direct sequence analysis showed a missense mutation of A for G substitution in the CYP 27 gene at codon 362 (CGT 362Arg to CAT 362His) with a homozygous pattern in the patient, and a heterozygous pattern in the parents. The mutation, which eliminates a normal HgaI endonuclease site at position 1195 of the cDNA and is located at the adrenodoxin binding region of the gene, is most probably responsible for the decreased sterol 27-hydroxylase activity in this Japanese CTX family. The combined data strongly support that the primary enzymatic defect in CTX is the disruption of sterol 27-hydroxylase and that the disease is inherited in an autosomal recessive trait.

Human evidence that the apolipoprotein a-II gene is implicated in visceral fat accumulation and metabolism of triglyceride-rich lipoproteins.

BACKGROUND: Apolipoprotein (apo) A-II is a major structural protein of plasma HDLs, but little is known regarding its functions. METHODS AND RESULTS: To investigate the physiological role of apoA-II in humans, we screened the promoter region of the apoA-II gene for a functional polymorphism and used this polymorphism as a tool in association studies. A common, functional polymorphism in the promoter region of the apoA-II gene, a T to C substitution at position -265, was found. Electrophoretic mobility shift assays demonstrated that the -265T/C polymorphism influences the binding of nuclear proteins, whereas transient transfection studies in human hepatoma cells showed a reduced basal rate of transcription of the -265C allele compared with the -265T allele. The -265C allele was associated with decreased plasma apoA-II concentration and decreased waist circumference in healthy 50-year-old men. In addition, oral fat tolerance tests provided evidence that the -265C allele enhances postprandial metabolism of large VLDLs. CONCLUSIONS: ApoA-II appears to promote visceral fat accumulation and impair metabolism of large VLDLs.

Isolation of tryptic fragments of human C4 expressing Chido and Rodgers antigens.

It has been shown previously that methylamine is incorporated into the alpha-chain of human C4, resulting in a loss of haemolytic function and the appearance of a free thiol group in the molecule. In the present study it was demonstrated that a fragment resembling C4d is liberated from C4 by trypsin. The fragment--Try-C4d--contains both the methylamine binding site and the free thiol group. When separated on DEAE-Sepharose, four types of Try-C4d, differing in charge and size, could be defined. The size difference was found to parallel the presence of Chido and Rodgers blood group antigens. Fragments of Mr 30,000 carried the Rodgers antigen and the Chido antigen was expressed on fragments of Mr 28,000.

Characterization of tryptic fragments of human complement factor C3.

C3c and C3d fragments were prepared in pure form from trypsin-digested human C3, and the individual chains of tryptic C3c were isolated by gel filtration on Sepharose 4B in 6M guanidinium hydrochloride. No low mol. wt (Mr) fragments were identified. The polypeptide chains were characterized with regard to Mr, amino acid composition and N-terminal amino acid sequence. Tryptic C3c consisted of one fragment from the beta-chain (Mr 64,000) and two from the alpha'-chain (Mr 40,000 and 23,000). The beta-chain fragment was derived from the C-terminal part of the chain, and the 23,000-Mr component constituted the amino terminal end of the alpha-chain. The 40,000-Mr fragment emanated from the C-terminal end of the alpha-chain. Tryptic C3d displayed microheterogeneity on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, but possessed a homogeneous N-terminal, identical to that described by Tack et al. (1980) (Proc. natn. Acad. Sci. U.S.A. 77, 5764-5768). By utilization of antisera against subunits of C3 and C3c in immunoblotting a degradation scheme for C3 by trypsin was proposed and the positions of the fragments in the intact molecule indicated.

Chemical characterization of cyanogen bromide fragments from the beta-chain of human complement factor C3.

The isolated beta-chain of human complement factor C3 (C3 beta) was fragmented by cyanogen bromide. Nine fragments were defined by gel filtration and high-pressure liquid chromatography, and characterized with respect to their Mr, amino acid composition and N-terminal amino acid sequence. Approx. 30% of the primary structure of C3 beta was determined. Alignment of the 3 N-terminal fragments allowed determination of 61 of the amino terminal residues of C3 beta. This region demonstrated 40% homology with the sequence in the N-terminal segment of the alpha-chain of the cobra venom factor.

Primary sequence differences between Chido and Rodgers variants of tryptic C4d of the human complement system.

Human tryptic C4d of the Chido and Rodgers variant was fragmented by cyanogen bromide and trypsin. The fragments were characterized by amino acid analysis and sequence determination. Polymorphism between the two genetic variants was detected in 5 positions. Four were closely located (residues 141, 142, 145, 146), where Leu, Ser, Ile, His occurred in the Chido variant and Pro, Cys, Leu, Asp in the Rodgers variant, respectively. In position 94 Gly was found in Chido and Asp in Rodgers. Alignment of the fragments was performed and it is concluded that tryptic C4d of both variants contains 346 residues.

Genetic characterization of Swedish patients with familial hypercholesterolemia: a heterogeneous pattern of mutations in the LDL receptor gene.

Familial hypercholesterolemia (FH) is an autosomal codominant disease, caused by mutations in the LDL receptor gene. To characterize the distribution of genetic aberrations in Swedish FH-patients fulfilling the clinical criteria of FH, we have investigated 150 unrelated Swedish patients for mutations in the LDL receptor gene and for the most common mutation causing familial ligand defective apo B-100 (FDB). Of the patients, 77 were recruited from Huddinge University Hospital in Stockholm and 73 from Sahlgren's University Hospital in Göteborg. Screening was carried out using SSCP and Southern blotting techniques, combined with DNA sequence analysis. In total, mutations regarded as cause for disease were identified in 55 patients (37%), representing 32 different types of mutations. In the LDL receptor gene we detected four nonsense mutations, 13 missense mutations, seven splice junction mutations, and four major rearrangements. In addition, two small deletions were identified and one base exchange in the promoter region. The most common mutation (apo B3500) causing FDB was found in three patients. The most frequent mutation was FH-Helsinki, reflecting the admixture of Finnish immigrants. We further identified 15 point mutations which were not considered to affect the function of the gene, and thus were regarded as polymorphic changes. This multitude of mutations reflects a heterogeneous genetic background in our series of Swedish FH-patients and differs from the situation in the other Scandinavian countries. Future studies should aim at characterizing the importance of other genes for the development of the FH phenotype.

Isolation of human complement factors C3, C5 and H.

An improved method for simultaneous purification of complement factors C3, C5 and H from human plasma has been developed. Using an initial batch separation technique with QAE-Sephadex, followed by chromatography on SP-Sephadex and gel filtration in Sephadex G-200, 600 mg of highly pure C3 can be prepared from 1600 ml of plasma. Simultaneously about 70 mg of highly pure factor H and 30 mg of C5 are obtained by chromatography of post SP-Sephadex material on DEAE-Sephacel. A small amount of C3 in the C5 pool is removed by anti-C3-Sepharose. By maleylation or citraconylation of reduced and alkylated C3, the constitutive polypeptide chains are modified in a way that made them separable by ion exchange chromatography.

Association between increased levels of reverse triiodothyronine and mortality after acute myocardial infarction.

PURPOSE: The thyroid hormone system may be downregulated temporarily in patients who are severely ill. This "euthyroid sick syndrome" may be an adaptive response to conserve energy. However, thyroid hormone also has beneficial effects on the cardiovascular system, such as improving cardiac function, reducing systemic vascular resistance, and lowering serum cholesterol levels. We investigated whether thyroid hormone levels obtained at the time of myocardial infarction are associated with subsequent mortality. PATIENTS AND METHODS: Serum levels of thyroid hormones (triiodothyronine [T3], reverse T3, free thyroxine [T4], and thyroid-stimulating hormone) were measured in 331 consecutive patients with acute myocardial infarction (mean age [+/- SD], 68 +/- 12 years), from samples obtained at the time of admission. RESULTS: Fifty-three patients (16%) died within 1 year. Ten percent (16 of 165) of patients with reverse T3 levels (an inactive metabolite) >0.41 nmol/L (the median value) died within the first week after myocardial infarction, compared with none of the 166 patients with lower levels (P <0.0004). After 1 year, the corresponding figures were 24% (40 of 165) versus 7.8% (13 of 166; P <0.0001). Reverse T3 levels >0.41 nmol/L were associated with an increased risk of 1-year mortality (hazard ratio = 3.0; 95% confidence interval: 1.4 to 6.3; P = 0.005), independent of age, previous myocardial infarction, prior angina, heart failure, serum creatinine level, and peak serum creatine kinase-MB fraction levels. CONCLUSION: Determination of reverse T3 levels may be a valuable and simple aid to improve identification of patients with myocardial infarction who are at high risk of subsequent mortality.

Complement deposition in renal allografts with early malfunction.

Among patients with early severe impairment of renal allograft function we have previously identified a group displaying isolated deposition of complement factor C3 in glomeruli. Here we studied the pattern of complement deposition more extensively in allograft biopsies from five patients using an immunofluorescence technique. We found a prominent deposition of C3c, C3d and C4d antigens in the glomerular capillary walls, and a positive reaction to vitronectin (S-protein), but only trace amounts of the complement factor C9 neoepitope. Clq, C4c, C3a, iC3b, factor B, properdin, immunoglobulins IgG, IgA or IgM were not found in glomeruli or in any other cortical structure. These findings indicate that most of the demonstrated glomerular C3 consists of C3b and/or C3c/C3d molecules. By immunoelectron microscopy the C3 antigen was found within the glomerular basement membrane. Our findings indicate that there is a mechanism of complement activation involving the early steps of the classical pathway, despite the lack of demonstrable immunoglobulins in the tissue. In analogy with similar reactions described recently in heart allografts, we suggest that this may be a manifestation of a humoral rejection, possibly mediated by a low titer of circulating antibodies directed against endothelial surface antigens, presumed to be the initial step leading to complement activation.

Lack of association between apolipoprotein E allele epsilon 4 and sporadic Alzheimer's disease.

Apolipoprotein E (apoE) is a protein involved in the transport of lipids and a component of Alzheimer's disease (AD) plaques. There are three common alleles of the apoE gene, designated epsilon 2, epsilon 3 and epsilon 4. An association between familial and sporadic AD and the epsilon 4 allele was recently reported. We have investigated Swedish Alzheimer patients and controls. The epsilon 4 allele frequency in familial and sporadic cases and in controls was 47, 22 and 18%, respectively. There was no significant difference between sporadic AD and controls but in familial cases the increased epsilon 4 allele frequency previously reported was confirmed.