Further characterization of human glucocorticoid receptor mutants, R477H and G679S, associated with primary generalized glucocorticoid resistance.

OBJECTIVE: Primary generalized glucocorticoid resistance is a rare condition characterized by a generalized insensitivity to glucocorticoids, to some extent due to an impaired function of the glucocorticoid receptor. Our earlier genetic analysis of the human glucocorticoid receptor (hGR) in 12 unrelated patients with primary generalized glucocorticoid resistance revealed two new mutations, R477H in exon 4 and G679S in exon 8 in two patients. In order to further study the molecular mechanisms underlying the phenotype of these mutations we have investigated their effect on glucocorticoid signal transduction. METHODS: We have studied the DNA-binding ability of the R477H mutant with an electrophoretic mobility shift assay (EMSA). The ability of the R477H and the G679S mutants to affect TNFα induced NF-κB activity and wild-type GR signalling was studied in transient transfection assays. RESULTS: In EMSA the R477H mutation showed a reduced ability to bind to a glucocorticoid-response element compared to the wild-type GR. In transient transfection assays both the R477H mutant and the G679S mutant showed a dominant negative effect on co-transfected wild-type GR in Cos 7 cells. However, both mutants showed full capacity to repress TNFα-induced NF-κB activity. CONCLUSION: The impaired DNA-binding of the hGR, R477H mutant may explain the severe phenotype of cortisol resistance seen with this mutation. The dominant negative effects of both mutants on wild-type GR signalling probably contribute to the patients' cortisol resistance.

Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice.

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.

Allele-specific regulation of MTTP expression influences the risk of ischemic heart disease.

Promoter polymorphisms in microsomal triglyceride transfer protein (MTTP) have been associated with decreased plasma lipids but an increased risk for ischemic heart disease (IHD), indicating that MTTP influences the susceptibility for IHD independent of plasma lipids. The objective of this study was to characterize the functional promoter polymorphism in MTTP predisposing to IHD and its underlying mechanism. Use of pyrosequencing technology revealed that presence of the minor alleles of the promoter polymorphisms -493G>T and -164T>C result in lower transcription of MTTP in vivo in the heart, liver, and macrophages. In vitro experiments indicated that the minor -164C allele mediates the lower gene expression and that C/EBP binds to the polymorphic region in an allele-specific manner. Furthermore, homozygous carriers of the -164C were found to have increased risk for IHD as shown in a case-control study including a total of 544 IHD patients and 544 healthy control subjects. We concluded that carriers of the minor -164C allele have lower expression of MTTP in the heart, mediated at least partly by the transcription factor CCAAT/enhancer binding protein, and that reduced concentration of MTTP in the myocardium may contribute to IHD upon ischemic damage.

Falsely Elevated Thyroid-Stimulating Hormone Results due to Interference by M-Component of IgG-Lambda Type.

Heterophilic antibodies but also M-components can interfere with laboratory tests causing erroneous results. We report the case of a 75-year-old man with myeloma and a monoclonal immunoglobulin component (M-component) that caused elevated thyroid-stimulating hormone (TSH) results. The M-component was of the IgG-lambda type. Thyroid markers were analyzed repeatedly, and there was a clear association between IgG concentrations and TSH values (R 2 = 0.724). The highest TSH value was 75 mIU/L. Polyethylene glycol (PEG) precipitation of intact immunoglobulins was used to investigate if there was an antibody-related interference problem. The PEG treatment normalized the TSH value, showing that the cause of the elevated TSH result was due to interference caused by the M-component. In conclusion, it is important to remember that both heterophilic antibodies and M-components may cause erroneous results.

Increased whole body energy expenditure and protection against diet-induced obesity in Cyp8b1-deficient mice is accompanied by altered adipose tissue features.

The aim of this study was to elucidate mechanisms whereby bile acids exert beneficial metabolic effects, using the Cyp8b1-/- mouse as model. These mice are unable to synthesize cholic acid, resulting in increased synthesis of chenodeoxycholic acid and enlarged bile acid pool. Cyp8b1-/- mice were found to be protected against high-fat diet induced obesity. Bomb calorimetry measurements showed increased faecal energy output in Cyp8b1-/ mice. Indirect calorimetry measurements demonstrated increased energy expenditure in Cyp8b1-/- mice. Meal tolerance tests revealed no differences in glucose disposal, but the insulin response was lower in Cyp8b1-/- mice. Intravenous glucose tolerance tests, as well as static incubations of isolated islets, showed no difference between the groups, whereas insulin tolerance tests demonstrated improved insulin sensitivity in Cyp8b1-/- mice. The genes encoding mitochondrial transcription factor A (TFAM) and type 2-iodothyronine deiodinase were upregulated in brown adipose tissue of Cyp8b1/- mice and Western blot analyses showed increased abundance of TFAM, and a trend towards increased abundance of UCP1. The upregulation of TFAM and UCP1 was accompanied by increased mitochondrial density, as shown by transmission electron microscopy. White adipocytes of Cyp8b1-/- mice exhibited increased responsiveness to both catecholamines and insulin in lipolysis experiments and increased insulin-stimulated lipogenesis. In conclusion, increased energy expenditure, mitochondrial density of brown adipocytes and faecal energy output may all contribute to the protection against diet-induced obesity of Cyp8b1-/- mice. Enhanced insulin sensitivity of Cyp8b1-/- mice is accompanied by increased hormonal responsiveness of white adipocytes.

Induction of blood-circulating bile acids supports recovery from myelosuppressive chemotherapy.

Chemotherapeutic agents can reduce bone marrow (BM) activity, causing myelosuppression, a common life-threatening complication of cancer treatment. It is challenging to predict the patients in whom prolonged myelosuppression will occur, resulting in a delay or discontinuation of the treatment protocol. An early indicator of recovery from myelosuppression would thus be highly beneficial in clinical settings. In this study, bile acids (BAs) were highly increased in the systemic circulation as a natural response during recovery from myelosuppression, supporting regeneration of BM cells. BA levels in the blood of pediatric cancer patients and mice treated with chemotherapeutic agents were increased, in synchrony with early proliferation of BM cells and recovery from myelosuppression. In a mouse model of altered BA composition, Cyp8b1 knockout mice, a subset of mice recovered poorly after chemotherapy. The poor recovery correlated with low levels and changes in composition of BAs in the liver and systemic circulation. Conversely, BA supplementation in chemotherapy-treated wild-type mice resulted in significantly improved recovery. The results suggest that part of the mechanism by which BAs support recovery is the suppression of endoplasmic reticulum stress pathways in expanding and recovering hematopoietic cells. The findings propose a novel role of BAs as early markers of recovery and active components of the recovery process after chemotherapy.

A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice).

BACKGROUND: Hyperbiliverdinaemia is a poorly defined clinical sign that has been infrequently reported in cases of liver cirrhosis or liver carcinoma, usually indicating a poor long-term prognosis. AIMS: To clarify the pathogenesis of hyperbiliverdinaemia in an extended case report. METHODS: A 64-year-old man with alcoholic cirrhosis was admitted to hospital with severe bleeding from oesophageal varices. Ultrasonography showed ascites, but no dilatation of the biliary tree. The skin, sclerae, plasma, urine and ascites of the patient showed a greenish appearance. Bilirubin levels were normal, and there were no signs of haemolysis. Biliverdin was analysed in plasma and urine with liquid chromatography coupled to mass spectrometry. The seven exonic regions of the biliverdin reductase-A (BVR-A) gene was amplified by polymerase chain reaction and sequenced. RESULTS: Biliverdin was present in plasma and urine. In nucleotide 52 of exon I of the DNA isolated from the hyperbiliverdinaemic patient, we discovered a novel heterozygous C-->T nonsense mutation converting an arginine (CGA) in position 18 into a stop codon (TGA) (R18Stop) predicted to truncate the protein N-terminally to the active site Tyr97. Two children of the proband were heterozygous for the identical mutation in the BVR-A gene, but had no clinical signs of liver disease and had normal levels of biliverdin. The BVR-A gene mutation was not found in 200 healthy volunteers or nine patients with end-stage liver cirrhosis. CONCLUSION: Hyperbiliverdinaemia (green jaundice) with green plasma and urine may be caused by a genetic defect in the BVR-A gene in conjunction with decompensated liver cirrhosis.

Promoter analysis of the murine squalene epoxidase gene. Identification of a 205 bp homing region regulated by both SREBP'S and NF-Y.

Squalene epoxidase (SE) is one of the most highly regulated enzymes of the cholesterol biosynthesis pathway. Here we identify the molecular basis for SREBP-2 synergy with NF-Y as the prime regulator of SE gene transcription. As expected cholesterol markedly suppressed transcriptional activity, while SREBP-1a, -1c and -2 activated it. Knock down of SREBP-2 mRNA resulted in an 85% reduction in SE expression. Interspecies comparison of SE promoter sequences identified two conserved putative NF-Y sites that were found to be important for maximal SREBP dependent gene activation and one novel conserved sterol response element (SRE). Altogether three novel SREs were identified within a 205 bp region of the SE promoter. Each of the SREs was capable of binding SREBP-2 but mutation of all three, singly or in combination, did not completely eliminate the SREBP response. Our results demonstrate the critical dependence of this 205 bp region for sterol dependent regulation of SE and uncover a possible framework for SREBP-promoter interaction, including a potent synergy with NF-Y that may be of principal importance.

Cholic acid mediates negative feedback regulation of bile acid synthesis in mice.

Cholesterol is converted into dozens of primary and secondary bile acids through pathways subject to negative feedback regulation mediated by the nuclear receptor farnesoid X receptor (FXR) and other effectors. Disruption of the sterol 12alpha-hydroxylase gene (Cyp8b1) in mice prevents the synthesis of cholate, a primary bile acid, and its metabolites. Feedback regulation of the rate-limiting biosynthetic enzyme cholesterol 7alpha-hydroxylase (CYP7A1) is lost in Cyp8b1(-/-) mice, causing expansion of the bile acid pool and alterations in cholesterol metabolism. Expression of other FXR target genes is unaltered in these mice. Cholate restores CYP7A1 regulation in vivo and in vitro. The results implicate cholate as an important negative regulator of bile acid synthesis and provide preliminary evidence for ligand-specific gene activation by a nuclear receptor.

Cholic acid as key regulator of cholesterol synthesis, intestinal absorption and hepatic storage in mice.

To study the effects of cholic acid (CA) feeding on hepatic cholesterol metabolism, male sterol 12alpha-hydroxylase (CYP8B1) knockout (-/-) mice and wildtype controls (+/+) were fed either a control diet or the same diet supplemented with CA (0.1% or 0.5% w/w) or cholesterol (1% w/w). During feeding of the control diet, cholesterol synthesis was increased in CYP8B1-/- compared to +/+ mice. Both cholesterol and CA feeding down regulated mRNA expression of cholesterogenic genes and hepatic de novo cholesterol synthesis as also reflected by a concomitant decrease in the nuclear factor SREBP-2 precursor protein and increased hepatic free cholesterol levels. Mice with an intact CYP8B1 gene (CYP8B1+/+ and C57Bl/6 mice) accumulated higher concentrations of cholesteryl esters (24- and 25-fold, respectively) in their livers compared to CYP8B1-/- mice (8-fold). Feeding of CA increased intestinal cholesterol absorption in CYP8B1+/+ mice by 23% and in CYP8B1-/- mice by 50%. While plasma cholesterol did not differ between CYP8B1+/+ and -/- mice under control conditions and cholesterol feeding a decrease was seen in CYP8B1-/- but not CYP8B1+/+ mice fed CA. This study indicates that CA is an important determinant for intestinal cholesterol absorption and that the levels of the transcription factor SREBP-2 in the liver are dependent upon the combined effect of CA on intestinal cholesterol absorption and CYP7A1. The possibility is discussed that inhibition of CYP8B1 and thus CA synthesis may be beneficial for the treatment of hyperlipidemic disorders.

Dexamethasone stimulates very low density lipoprotein (VLDL) receptor gene expression in differentiating 3T3-L1 cells.

To characterize endocrine mechanisms of very low density lipoprotein (VLDL) receptor regulation we studied mouse adipocytic 3T3-L1 cells. Lipid filled adipocyte-like cells are formed during a 5-7 day time course in the presence of insulin, dexamethasone and isobutylmethylxanthine (IBMX). The VLDL receptor protein, in the form of its approximately 120 and approximately 100 kDa type I and type II isoforms, as well as binding of (125)I-beta-VLDL, was induced several-fold during differentiation. Among the three different constituents added to the culture medium only dexamethasone (1 microM), but not insulin or IBMX, induced a time- and dose-dependent increase of VLDL receptor expression. Inclusion of RU-486 (10 microM) blocked the stimulatory effect of dexamethasone on VLDL receptor mRNA and protein levels. 3.6 kb of the 5'-untranslated region representing the VLDL receptor promoter were cloned and sequenced, and the transcriptional start site was determined by primer extension to be located 574 bases upstream from the initiating methionine. To investigate the functionality of the promoter, luciferase reporter gene constructs for the region -181 to -3726 bases were assembled and transfected into 3T3-L1 cells. An increased reporter gene activity was recorded when comparing preconfluent cells to fully differentiated cells. Between day 0 and day 2 (48 h after transfection) reporter gene activity was induced by dexamethasone, but not by insulin or IBMX. RU-486 inhibited this stimulatory effect for all constructs tested. No classical glucocorticoid receptor (GR) response element was found in the sequenced region of the VLDL receptor promoter. Thus, an indirect stimulatory effect mediated via GR on VLDL receptor gene transcription is the most likely mechanism of VLDL receptor gene activation in differentiating 3T3-L1 cells.

LXR driven induction of HDL-cholesterol is independent of intestinal cholesterol absorption and ABCA1 protein expression.

We investigated whether: (1) liver X receptor (LXR)-driven induction of high-density lipoprotein cholesterol (HDL-C) and other LXR-mediated effects on cholesterol metabolism depend on intestinal cholesterol absorption; and (2) combined treatment with the LXR agonist GW3965 and the cholesterol absorption inhibitor ezetimibe results in synergistic effects on cholesterol metabolism that could be beneficial for treatment of atherosclerosis. Mice were fed 0.2 % cholesterol and treated with GW3965+ezetimibe, GW3965 or ezetimibe. GW3965+ezetimibe treatment elevated serum HDL-C and Apolipoprotein (Apo) AI, effectively reduced the intestinal cholesterol absorption and increased the excretion of faecal neutral sterols. No changes in intestinal ATP-binding cassette (ABC) A1 or ABCG5 protein expression were observed, despite increased mRNA expression, while hepatic ABCA1 was slightly reduced. The combined treatment caused a pronounced down-regulation of intestinal Niemann-Pick C1-like 1 (NPC1L1) and reduced hepatic and intestinal cholesterol levels. GW3965 did not affect the intestinal cholesterol absorption, but increased serum HDL-C and ApoAI levels. GW3965 also increased Apoa1 mRNA levels in primary mouse hepatocytes and HEPA1-6 cells. Ezetimibe reduced the intestinal cholesterol absorption, ABCA1 and ABCG5, but did not affect the serum HDL-C or ApoAI levels. Thus, the LXR-driven induction of HDL-C and ApoAI was independent of the intestinal cholesterol absorption and increased expression of intestinal or hepatic ABCA1 was not required. Inhibited influx of cholesterol via NPC1L1 and/or low levels of intracellular cholesterol prevented post-transcriptional expression of intestinal ABCA1 and ABCG5, despite increased mRNA levels. Combined LXR activation and blocked intestinal cholesterol absorption induced effective faecal elimination of cholesterol.

Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect ß-oxidation.

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial ß-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect ß-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

A functional polymorphism in the HMGCR promoter affects transcriptional activity but not the risk for Alzheimer disease in Swedish populations.

Variations in genes associated with cholesterol homeostasis have been reported to modify the risk of developing Alzheimer disease (AD). To date there have been few investigations into variations in genes directly involved in cholesterol biosynthesis and AD. We investigated the influence of the -911C>A polymorphism (rs3761740) in the hydroxy-methyl-glutaryl CoA reductase (HMGCR) gene promoter on basal and regulated transcription, plasma cholesterol levels and the association with AD. Under in vitro conditions the A allele was found to be significantly more responsive to SREBP-2 mediated regulation than the C allele. In an age and sex matched case-control study, the genotype distribution and allele frequency of this polymorphism were not associated with AD (OR=1.03; 95% CI=0.72-1.48). However, we did find evidence supporting an interaction between the HMGCR A allele, the APOE E4 allele and an altered risk of AD (OR=2.41; 95% CI=0.93-6.22).

Regulation by SREBP-2 defines a potential link between isoprenoid and adenosylcobalamin metabolism.

Mevalonate kinase (MVK) catalyses an early step in cholesterol biosynthesis converting mevalonate to phosphomevalonate. Cob(I)alamin adenosyltransferase (MMAB) converts cob(I)alamin to adenosylcobalamin, functionally required for mitochondrial methylmalonyl-CoA mutase activity and succinyl-CoA formation. These two synthenic genes are found in a head-to-head formation on chromosome 12 in man and chromosome 5 in mouse. The 330bp intergenic region showed several conserved NF-Y sites indicative of potential bidirectional regulatory SREBP synergism. Both MVK and MMAB appear to be regulated in a similar manner, to a large extent by SREBP-2, since their tissue expression pattern was similar and both genes were suppressed by an excess of cholesterol as well as SREBP-2 knockdown. Statin treatment in mice upregulated both Mvk and Mmab mRNA levels indicating that this treatment may be useful in inborn errors of cblB complementation associated with methylmalonic aciduria as well as hyper IgD and periodic fever syndrome (HIDS).

Differential hepatocellular zonation pattern of cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1) in the mouse.

The synthesis of primary bile acids is confined to the hepatocytes. This study aimed to evaluate the expression pattern within the liver architecture of the rate-limiting enzyme of the neutral pathway, cholesterol 7alpha-hydroxylase (Cyp7a1), and sterol 12alpha-hydroxylase (Cyp8b1), the enzyme necessary for the synthesis of cholic acid. Specific Cyp8b1 and Cyp7a1 peptide antiserums were used for immunohistochemical staining of livers from wild type and Cyp8b1 null mice, the latter instead expressing beta-galactosidase (beta-Gal) as a replacement reporter gene. Cyp8b1 was mainly expressed in the hepatocytes in a zonal pattern surrounding the central vein while the areas surrounding the portal zones showed much lower levels. The zonation was maintained in cholic acid-depleted mice using beta-Gal as a reporter protein. Cyp7a1 expression in wild type mice also showed a zonal distribution pattern, although less distinct, with a maximal expression within a 1-2 cell thick layer of hepatocytes surrounding the central vein. In Cyp8b1 null mice, a more intense staining was obtained, in accordance with the higher expression level of Cyp7a1, although the overall expression pattern was maintained. Our results in mice indicate possible differences in the regulation of the cellular zonation of Cyp7a1 and Cyp8b1. Also, cholic acid affects the set-point of Cyp7a1 expression but not its zonal distribution.

Critical role of cholic acid for development of hypercholesterolemia and gallstones in diabetic mice.

We studied bile acid and cholesterol metabolism in insulin-dependent diabetes utilizing genetically modified mice unable to synthesize cholic acid (Cyp8b1-/-). Diabetes was induced in Cyp8b1-/- and wild type animals (Cyp8b1+/+) by alloxan, and the mice were fed normal or cholesterol-enriched diet for 10 weeks. The serum levels of cholesterol were strongly increased in diabetic Cyp8b1+/+ mice fed cholesterol, while diabetic Cyp8b1-/- mice did not show any aberrations regardless of the diet. Diabetic cholesterol-fed Cyp8b1+/+ mice had much higher biliary cholesterol and cholesterol saturation index than all other groups, their bile contained a large number of cholesterol crystals, and their canalicular cholesterol transporter Abcg5/g8 mRNA levels were much higher. Cyp7a1 mRNA levels were similar in all diabetic mice but higher compared to non-diabetic animals. The results indicate a critical role for cholic acid for the development of hypercholesterolemia and gallstones in our animal model.

Expression of iron regulatory genes in a rat model of hepatocellular carcinoma.

BACKGROUND/AIMS: The altered iron metabolism in hepatocellular carcinomas (HCCs), characterized by the iron-deficient phenotype, is suggested to be of importance for tumour growth. However, the underlying molecular mechanisms remain poorly understood. We asked whether these iron perturbations would involve altered expression of genes controlling iron homeostasis. METHODS: HCCs were induced in rats by the Solt and Farber protocol of chemical hepatocarcinogenesis, and to evaluate the effects of iron loading, one group of animals were supplemented with dietary iron during tumour progression. Tissue iron contents were determined, labelling indices of S-phase nuclei were calculated, and mRNA levels of iron-regulatory genes were quantitated. Protein levels of ferroportin1 were determined with Western blot. RESULTS: HCCs displayed reduced amount of tissue iron and lack of histologically stainable iron. HCCs expressed significantly higher mRNA levels of genes involved in iron uptake (transferrin receptor-1, divalent metal ion transporter-1), ferroxidase activity (Ferritin-H), and iron extrusion (ferroportin1). The protein levels of ferroportin1 in iron-deficient HCCs were similar as in control livers, and did not increase in HCCs exposed to iron. Hepcidin mRNA levels were decreased in iron-deficient HCCs, rose in response to iron loading and correlated to the tissue iron content. CONCLUSIONS: Taken together, the altered expressions of iron-regulatory genes in HCCs possibly reflect an increased demand for bioavailable iron and a high iron turnover in neoplastic cells.