Editor's Choice - Nationwide Analysis of Patients Undergoing Iliac Artery Aneurysm Repair in the Netherlands.

OBJECTIVE: The new 2019 guideline of the European Society for Vascular Surgery (ESVS) recommends consideration for elective iliac artery aneurysm (eIAA) repair when the iliac diameter exceeds 3.5 cm, as opposed to 3.0 cm previously. The current study assessed diameters at time of eIAA repair and ruptured IAA (rIAA) repair and compared clinical outcomes after open surgical repair (OSR) and endovascular aneurysm repair (EVAR). METHODS: This retrospective observational study used the nationwide Dutch Surgical Aneurysm Audit (DSAA) registry that includes all patients who undergo aorto-iliac aneurysm repair in the Netherlands. All patients who underwent primary IAA repair between 1 January 2014 and 1 January 2018 were included. Diameters at time of eIAA and rIAA repair were compared in a descriptive fashion. The anatomical location of the IAA was not registered in the registry. Patient characteristics and outcomes of OSR and EVAR were compared with appropriate statistical tests. RESULTS: The DSAA registry comprised 974 patients who underwent IAA repair. A total of 851 patients were included after exclusion of patients undergoing revision surgery and patients with missing essential variables. eIAA repair was carried out in 713 patients, rIAA repair in 102, and symptomatic IAA repair in 36. OSR was performed in 205, EVAR in 618, and hybrid repairs and conversions in 28. The median maximum IAA diameter at the time of eIAA and rIAA repair was 43 (IQR 38-50) mm and 68 (IQR 58-85) mm, respectively. Mortality was 1.3% (95% CI 0.7-2.4) after eIAA repair and 25.5% (95% CI 18.0-34.7) after rIAA repair. Mortality was not significantly different between the OSR and EVAR subgroups. Elective OSR was associated with significantly more complications than EVAR (intra-operative: 9.8% vs. 3.6%, post-operative: 34.0% vs. 13.8%, respectively). CONCLUSION: In the Netherlands, most eIAA repairs are performed at diameters larger than recommended by the ESVS guideline. These findings appear to support the recent increase in the threshold diameter for eIAA repair.

Endoscopic sphincterotomy and cholecystectomy in acute biliary pancreatitis.

BACKGROUND: This review discusses current insights with regard to biliary tract management during and after acute biliary pancreatitis. METHODS: A MEDLINE and EMBASE search was done and studies were selected based on methodological quality and publication date. The recommendations of recent guidelines are incorporated in this review. In absence of consensus in the literature, expert opinion is expressed. RESULTS: There is no role for early endoscopic retrograde cholangiopancreatography (ERCP) in patients with (predicted) mild biliary pancreatitis to improve outcome. In case of persisting choledocholithiasis, ERCP with stone extraction is scheduled electively when the acute event has subsided. Whether early ERCP with sphincterotomy is beneficial in patients with predicted severe pancreatitis remains subject to debate. Regardless of disease severity, in case of concomitant cholangitis urgent endoscopic sphincterotomy (ES) is recommended. As a definitive treatment to reduce the risk of recurrent biliary events in the long term, ES is inferior to cholecystectomy and should be reserved for patients considered unfit for surgery. After severe biliary pancreatitis, cholecystectomy should be postponed until all signs of inflammation have subsided. In patients with mild pancreatitis, cholecystectomy during the primary admission reduces the risk of recurrent biliary complications. CONCLUSION: Recent research has provided valuable data to guide biliary tract management in the setting of acute biliary pancreatitis with great value and benefit for patients and clinicians. Some important clinical dilemmas remain, but it is anticipated that on-going clinical trials will deliver some important insights and additional guidance soon.

Staged multidisciplinary step-up management for necrotizing pancreatitis.

BACKGROUND: Some 15 per cent of all patients with acute pancreatitis develop necrotizing pancreatitis, with potentially significant consequences for both patients and healthcare services. METHODS: This review summarizes the latest insights into the surgical and medical management of necrotizing pancreatitis. General management strategies for the treatment of complications are discussed in relation to the stage of the disease. RESULTS: Frequent clinical evaluation of the patient's condition remains paramount in the first 24-72 h of the disease. Liberal goal-directed fluid resuscitation and early enteral nutrition should be provided. Urgent endoscopic retrograde cholangiopancreatography is indicated when cholangitis is suspected, but it is unclear whether this is appropriate in patients with predicted severe biliary pancreatitis without cholangitis. Antibiotic prophylaxis does not prevent infection of necrosis and antibiotics are not indicated as part of initial management. Bacteriologically confirmed infections should receive targeted antibiotics. With the more conservative approach to necrotizing pancreatitis currently advocated, fine-needle aspiration culture of pancreatic or extrapancreatic necrosis will less often lead to a change in management and is therefore indicated less frequently. Optimal treatment of infected necrotizing pancreatitis consists of a staged multidisciplinary 'step-up' approach. The initial step is drainage, either percutaneous or transluminal, followed by surgical or endoscopic transluminal debridement only if needed. Debridement is delayed until the acute necrotic collection has become 'walled-off'. CONCLUSION: Outcome following necrotizing pancreatitis has improved substantially in recent years as a result of a shift from early surgical debridement to a staged, minimally invasive, multidisciplinary, step-up approach.

Systematic review of percutaneous catheter drainage as primary treatment for necrotizing pancreatitis.

BACKGROUND: The role of percutaneous catheter drainage (PCD) in patients with (infected) necrotizing pancreatitis was evaluated. METHODS: A systematic literature search was performed. Inclusion criteria were: consecutive cohort of patients with necrotizing pancreatitis undergoing PCD as primary treatment for peripancreatic collections; indication for PCD either (suspected) infected necrosis or symptomatic sterile pancreatic necrosis; and outcomes reported to include percentage of infected peripancreatic collections, need for additional surgical necrosectomy, complications and deaths. Exclusion criteria were: cohort of fewer than five patients; cohort included patients with chronic pancreatitis; selected subgroup of patients with acute pancreatitis studied, such as those with pseudocysts, pancreatic abscesses and/or exclusively sterile pancreatic necrosis; and cohort in which PCD was combined with another minimally invasive strategy and results for PCD alone not reported separately. RESULTS: Eleven studies, including 384 patients, fulfilled the inclusion criteria. Only one study was a randomized controlled trial; most others were retrospective case series. Four studies reported on the presence of organ failure before PCD; this occurred in 67·2 per cent of 116 patients. Infected necrosis was proven in 271 (70·6 per cent) of 384 patients. No additional surgical necrosectomy was required after PCD in 214 (55·7 per cent) of 384 patients. Complications consisted mostly of internal and external pancreatic fistulas. The overall mortality rate was 17·4 per cent (67 of 384 patients). Nine of 11 studies reported mortality separately for patients with infected necrosis undergoing PCD; the mortality rate in this group was 15·4 per cent (27 of 175). CONCLUSION: A considerable number of patients can be treated with PCD without the need for surgical necrosectomy.

Timing of cholecystectomy after mild biliary pancreatitis.

BACKGROUND: The aim of the study was to evaluate recurrent biliary events as a consequence of delay in cholecystectomy following mild biliary pancreatitis. METHODS: Between 2004 and 2007, patients with acute pancreatitis were registered prospectively in 15 Dutch hospitals. Patients with mild biliary pancreatitis were candidates for cholecystectomy. Recurrent biliary events requiring admission before and after cholecystectomy, and after endoscopic sphincterotomy (ES), were evaluated. RESULTS: Of 308 patients with mild biliary pancreatitis, 267 were candidates for cholecystectomy. Eighteen patients underwent cholecystectomy during the initial admission, leaving 249 potential candidates for cholecystectomy after discharge. Cholecystectomy was performed after a median of 6 weeks in 188 patients (75·5 per cent). Before cholecystectomy, 34 patients (13·7 per cent) were readmitted for biliary events, including 24 with recurrent biliary pancreatitis. ES was performed in 108 patients during the initial admission. Eight (7·4 per cent) of these patients suffered from biliary events after ES and before cholecystectomy, compared with 26 (18·4 per cent) of 141 patients who did not have ES (risk ratio 0·51, 95 per cent confidence interval 0·27 to 0·94; P = 0·015). Following cholecystectomy, eight (3·9 per cent) of 206 patients developed biliary events after a median of 31 weeks. Only 142 (53·2 per cent) of 267 patients were treated in accordance with the Dutch guideline, which recommends cholecystectomy or ES during the index admission or within 3 weeks thereafter. CONCLUSION: A delay in cholecystectomy after mild biliary pancreatitis carries a substantial risk of recurrent biliary events. ES reduces the risk of recurrent pancreatitis but not of other biliary events.

Prediction of common bile duct stones in the earliest stages of acute biliary pancreatitis.

BACKGROUND AND STUDY AIMS: Accurate prediction of common bile duct (CBD) stones in acute biliary pancreatitis is warranted to select patients for early therapeutic endoscopic retrograde cholangiopancreatography (ERCP). We evaluated commonly used biochemical and radiological predictors of CBD stones in a large prospective cohort of patients with acute biliary pancreatitis who were undergoing early ERCP. PATIENTS AND METHODS: 167 patients with acute biliary pancreatitis who were undergoing early ERCP (< 72 hours after symptom onset) in 15 Dutch hospitals in 2004 - 2007 were prospectively included. Abdominal ultrasonography and/or computed tomography (CT) was performed on admission and complete liver biochemistry determined daily. We used univariate logistic regression to assess associations between CBD stones found during ERCP (gold standard) and the following parameters: (1) clinical: age, sex, predicted severity; (2) radiological: dilated CBD, impacted stone in CBD; and (3) biochemical: bilirubin, γ-glutamyltransferase (GGT), alkaline phosphatase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). RESULTS: Out of 167 patients, 94 (56 %) had predicted severe acute biliary pancreatitis, 51 (31%) exhibited a dilated CBD and 15 (9%) had CBD stones on ultrasonography and/or CT. ERCP was performed at a median of 0 days (interquartile range 0 - 1) after admission. CBD stones were found during ERCP in 89/167 patients (53%). In univariate analysis, the only parameters significantly associated with CBD stones were GGT (per 10 units increase: odds ratio 1.02, 95% CI 1.01 - 1.03, P = 0.001) and alkaline phosphatase (per 10 units increase: odds ratio 1.03, 95% CI 1.00 - 1.05, P = 0.028). These and all other tested parameters, however, showed poor positive predictive value (ranging from 0.53 to 0.69) and poor negative predictive value (ranging from 0.46 to 0.67). CONCLUSIONS: The results of this study suggest that commonly used biochemical and radiological predictors of the presence of CBD stones during ERCP in the earliest stages of acute biliary pancreatitis are unreliable.

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as 'fold-difference' results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.

A practical view of fine-mapping and gene prioritization in the post-genome-wide association era.

Over the past 15 years, genome-wide association studies (GWASs) have enabled the systematic identification of genetic loci associated with traits and diseases. However, due to resolution issues and methodological limitations, the true causal variants and genes associated with traits remain difficult to identify. In this post-GWAS era, many biological and computational fine-mapping approaches now aim to solve these issues. Here, we review fine-mapping and gene prioritization approaches that, when combined, will improve the understanding of the underlying mechanisms of complex traits and diseases. Fine-mapping of genetic variants has become increasingly sophisticated: initially, variants were simply overlapped with functional elements, but now the impact of variants on regulatory activity and direct variant-gene 3D interactions can be identified. Moreover, gene manipulation by CRISPR/Cas9, the identification of expression quantitative trait loci and the use of co-expression networks have all increased our understanding of the genes and pathways affected by GWAS loci. However, despite this progress, limitations including the lack of cell-type- and disease-specific data and the ever-increasing complexity of polygenic models of traits pose serious challenges. Indeed, the combination of fine-mapping and gene prioritization by statistical, functional and population-based strategies will be necessary to truly understand how GWAS loci contribute to complex traits and diseases.

IL-1B -31T>C promoter polymorphism is associated with gastric stump cancer but not with early onset or conventional gastric cancers.

It has been reported that interleukin-1beta (IL-1B) genes play a crucial role in the genetic predisposition to gastric cancer although there is no information about their role in different subtypes of gastric cancer. We performed single nucleotide polymorphism analysis of IL-1B in 241 gastric cancers including early onset gastric cancers (EOGC), conventional gastric cancers, and gastric stump cancers (GSCs) as well as 100 control patients, using real-time polymerase chain reaction and sequence analysis. The C allele was present in 60% of EOGCs, 59% of conventional gastric cancers, and 90% of GSCs, compared to 62% in the control group. Interestingly, there was no difference between early onset and conventional gastric cancer with respect to the IL-1B -31T>C polymorphism distribution. A statistically significant difference in the presence of the C allele compared to the control group was found in patients with gastric stump cancer (p = 0.008) with the T allele conferring protection against gastric stump cancer. In summary, we have shown that the IL-1B -31C allele promoter polymorphism is significantly associated with gastric stump cancer compared to the control group. Although several molecular differences have been identified between conventional gastric cancer and early onset gastric cancer, the IL-1B -31 allele distribution is similar between these two groups.

Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components.

OBJECTIVE: In view of their different actions on thyroid hormone receptor (TR) isoforms we set out to investigate whether amiodarone (AM) and dronedarone (Dron) have different and/or component-specific effects on cardiac gene expression. DESIGN: Rats were treated with AM or Dron and the expression of TRalpha 1, TRalpha 2, TRbeta 1 and several tri-iodothyronine (T3)-regulated genes was studied in different parts of the heart, namely the right atrium (RA), left ventricular wall (LVW) and apex. METHODS: Rats were treated for 14 days with 100 mg/kg body weight AM or Dron. The expression of TRalpha 1, TRalpha 2, TRbeta 1 and T3-regulated genes was studied using real-time PCR and non-radioactive in situ hybridisation. RESULTS: AM and Dron affected TR expression in the RA similarly by decreasing TRalpha 1 and beta 1 expression by about 50%. In the LVW, AM and Dron decreased TRbeta 1 and, interestingly, AM increased TRalpha 1. In the apex, AM also increased TRalpha 2. The changes seen in T3-dependent gene expression are reminiscent of foetal reprogramming. CONCLUSION: Taken together, our results indicate that AM and Dron have similar effects on the expression of TR isoforms in the RA, which could partly contribute to their ability to decrease heart rate. On the other hand, the more profound effect of AM appears on TR- and T3-dependent gene expression in the left ventricle suggests foetal reprogramming.

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region.

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

PGC-1alpha regulates the isoform mRNA ratio of the alternatively spliced thyroid hormone receptor alpha transcript.

Transcripts derived from the thyroid hormone receptor alpha (TRalpha) gene are alternatively spliced resulting in a functional receptor TRalpha1 and a non-T3-binding variant TRalpha2 that can exert a dominant negative effect on the transactivation functions of other TRs. There is evidence that the ratio of TRalpha isoform transcripts can be modulated and here, we investigate whether the PPARgamma co-activator alpha (PGC-1alpha) has an effect on this splicing process. PGC-1alpha was discovered not only as a transcriptional co-activator, but also has certain motifs characteristic of splicing factors. We demonstrate that PGC-1alpha alters the ratio of endogenously expressed TRalpha isoform transcripts in HepG2 cells, by decreasing TRalpha1 mRNA levels twofold. This change in isoform ratio is accompanied by a decrease in 5'-deiodinase expression, whereas no differences were found in TRbeta1 expression. Deletion of the RNA-processing domain of PGC-1alpha abrogated the effect on the TRalpha splicing, whereas expression of only the RNA-processing domain favored TRalpha1 expression. PGC-1alpha showed a similar effect on the splicing of a TRalpha minigene containing only the last four exons and introns of the TRalpha gene. These data suggest that PGC-1alpha is involved in the RNA processing of TRalpha transcripts.

Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart.

Nuclear thyroid hormone (T3) receptors (TR) play a critical role in mediating the effects of T3 on development, differentiation and normal physiology of many organs. The heart is a major target organ of T3, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRalpha1, TRalpha2 and TRbeta1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRalpha1, TRalpha2 and TRbeta1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRbeta1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRalpha1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRalpha1 and TRbeta1 isoforms are co-expressed. We also found the heterogeneous expression of the TRalpha1, TRalpha2 and TRbeta1 isoforms in the developing mouse heart, which, in the case of the TRbeta1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRbeta1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRalpha1 and TRbeta1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.

Functional thyrotropin receptor expression in the pituitary folliculo-stellate cell line TtT/GF.

Thyrotropin secretion from the anterior pituitary is regulated mainly through TRH and thyroid hormones. Recent findings of a TSH receptor (TSHR) on folliculo-stellate (FS) cells in the human anterior pituitary indicate that TSH secretion might, in addition, be regulated in a paracrine manner via FS cells. In order to elucidate the physiological relevance of TSHR expression in FS cells we evaluated the effects of TSH on a murine FS cell line, TtT/GF. First, Western blot analysis confirmed the expression of TSHR protein in these cells. Second, three potential second messenger pathways were studied. Last, cDNA array hybridization was used to evaluate the effect of TSH on gene expression levels. TSH failed to induce either the adenylate cyclase/cAMP pathway, the phosphatidylinositol/calcium pathway, or the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway. Most of the genes regulated by TSH were related to cell proliferation, cell differentiation, and apoptosis. Moreover, TSH induced STAT5a and TGFbeta2 expression. We report that TtT/GF cells express a functional TSHR that is not coupled to cAMP nor IP (3) but probably signals through the JAK/STAT5a pathway. Functional TSHR expression in this cell line offers an in vitro model to study the role of TSHR in FS cells.

Tissue-specific differences in the binding of nuclear proteins to a CCAAT motif in the promoter of the androgen-regulated C3 gene.

The expression of the gene for the C3 polypeptide is confined to the ventral prostate of the male rat and is regulated by androgens. To study the mechanism of this tissue-specific and hormone-dependent regulation we have used DNase-I footprinting and band-shift assays to locate binding sites for nuclear proteins isolated from different tissues. In this paper we present evidence that there are tissue-specific differences in the nuclear proteins that are able to bind to a CCAAT motif and a nuclear factor-I consensus that are present in the promoter of the rat C3 gene. Using competition assays and heat denaturation we show that the CAAT box/enhancer binding protein itself may be one of the transcription factors involved.

The decrease of liver LDL receptor mRNA during fasting is related to the decrease in serum T3.

UNLABELLED: Fasting is associated with a reduction in serum T3 and T4 and a rise of plasma LDL cholesterol. We hypothesized that an hypothyroid-like condition induced by fasting is responsible for the rise in LDL cholesterol. We therefore examined the relation between changes in thyroid hormone and cholesterol metabolism in rats fasted for 0, 8, 12, 24 or 48 h. Fasting resulted in a decrease of liver 5'-deiodinase mRNA from 8 h (to 50%, p < 0.05, n = 6), of serum T3 from 12 h and of serum T4 at 48 h; serum TSH remained unchanged. Furthermore, plasma LDL cholesterol increased from 24 h onwards preceded by a decrease of liver LDL receptor mRNA which in turn is related to serum T3 (r = 0.55, p < 0.05, n = 19). Adding T3 at a concentration such that normal T3 levels are maintained during 48 h fasting, prevents the decrease in the LDL receptor mRNA. Fasting did not change hepatic HMG CoA reductase mRNA but decreased cholesterol 7 alpha-hydroxylase mRNA, which however was not related to the decrease of serum T3. IN CONCLUSION: (1) Fasting induces a hypothyroid-like condition in which inhibition of hepatic conversion of T4 into T3 may be responsible for the decrease of serum T3. (2) Fasting induces an increase of plasma LDL cholesterol, apparently caused by a decrease of hepatic LDL receptor gene expression which is (partly) related to the fall in serum T3.

Desethylamiodarone is a noncompetitive inhibitor of the binding of thyroid hormone to the thyroid hormone beta 1-receptor protein.

It has been hypothesized that amiodarone (A), a potent antiarrythmic and antianginal drug, induces a local hypothyroid-like condition in extrathyroidal tissues. This might be related to competitive antagonism of A for the thyroid hormone receptor reported in some studies but denied in others. These conflicting results are presumably due to the poor solubility of A in a hydrophilic environment. We, therefore, studied the effect of the drug and its major metabolite, desethylamiodarone (DEA), on the in vitro binding of thyroid hormone (T3) to its receptor protein using the rat beta 1-thyroid hormone receptor expressed in Escherichia coli. A and DEA stayed in solution up to 10(-4) M when 0.05% Triton X-100 was added to the incubation buffer, as evidenced by a recovery of 80-90% for both chemicals, as measured by HPLC. DEA, but not A, had a clear inhibitory effect on the binding of T3 to its receptor (IC50, 1-3 x 10(-5) M). Scatchard analysis in the presence of DEA demonstrated a dose-dependent decrease in the Ka as well as the maximum binding capacity. Lineweaver-Burke analysis indicated noncompetitive inhibition. Plots of the intercepts of Lineweaver-Burke plots vs. DEA concentration were linear (y = 0.334 + 0.098x), giving a Ki of 30 microM for the binding of DEA to the occupied receptor. Plots of the slopes vs. inhibitor concentration were parabolic (y = 3.01 + 0.06x + 0.16x2), indicating a progressively stronger effect of DEA on the unoccupied receptor as concentrations rise. This preference for the unoccupied receptor is reflected in experiments that show a progressive loss of T3 binding when the receptor was incubated for increasing periods with DEA before adding T3. We conclude that DEA is a noncompetitive inhibitor of the binding of T3 to the beta 1-thyroid hormone receptor protein, interacting preferably with the unoccupied T3 receptor.

Structure-function relationship of the inhibition of the 3,5,3'-triiodothyronine binding to the alpha1- and beta1-thyroid hormone receptor by amiodarone analogs.

Desethylamiodarone (DEA), the major metabolite of the potent antiarrhythmic drug amiodarone (A), acts as a competitive inhibitor of T3, binding to the alpha1-thyroid hormone receptor (alpha1-T3R), but as a noncompetitive inhibitor with respect to the beta1-T3R. To gain insight into the structure- function relationship of the interaction between A metabolites and T3Rs, we investigated the effects of several A analogs on T3 binding to the alpha1-T3R and beta1-T3R in vitro. The analogs tested were: 1) compounds obtained by deethylation of A, DEA, and desdiethylamiodarone (DDEA); 2) compounds obtained by deiodination of A, monoiodoamiodarone and desdiiodoamiodarone (DDIA); and 3) benzofuran derivatives with various iodination grades, 2-butyl-3-(4-hydroxy-3,5-diiodo-benzoyl)benzofuran (L3373, two iodine atoms), L6424 (L3373 with one iodine atom), and L3372 (L3373, no iodine atoms). IC50, values of inhibition of T3 binding to alpha1-T3R and beta1-T3R, respectively, were as follows (mean +/- SD, expressed x 10(-5) M): DEA, 4.7 +/- 0.9 and 2.7 +/- 1.4 (P < 0.001); DDEA, 3.7 +/- 0.9 and 1.9 +/- 0.3 (P < 0.001); monoiodoamiodarone, more than 20 and more than 20; DDIA, 16.2 +/- 5.6 and 9.1 +/- 2.1 (P < 0.01); L3373, 3.8 +/- 1.0 and 3.6 +/- 0.5 (P = NS); L6424, 11.3 +/- 5.7 and 10 +/- 2.0 (P = NS); and L3372, no inhibition. Scatchard analyses in the presence of DDEA, DDIA, and L3373 demonstrated a dose-dependent decrease in Ka, but no change in the maximum binding capacity (MBC) of T3 binding to alpha1-T3R. Langmuir plots clearly indicated competitive inhibition of T3 binding to alpha1-T3R by DDEA, DDIA, and L3373. In contrast, these three analogs acted differently with respect to the beta1-T3R. DDEA and DDIA decreased both Ka and MBC in Scatchard plots using beta1-T3R, demonstrating noncompetitive inhibition. L3373 decreased dose-dependently Ka, but not MBC, values of T3 binding to the beta1-T3R and clearly acted as a competitive inhibitor. Ki plots indicated that DDEA, DDIA, and L3373 do not interfere significantly with occupied T3Rs. KI (inhibition constant for the unoccupied receptor) plots demonstrated increasing inhibition of the T3 binding to unoccupied receptors with increasing analog concentrations. In summary, 1) removal of one or two ethyl groups of A results in compounds with strong but almost equal potency of inhibiting T3R binding, whereas removal of one or two iodine atoms of A has a lower potency in this respect. The strong inhibitory potency of the benzofuran derivative L3373 (equalling that of the deethylated compounds) is lost upon deiodination. 2) All tested A analogs acted as competitive inhibitors to the alpha1-T3R. The behavior to the beta1-T3R was different; deethylation or deiodination of A resulted in noncompetitive inhibition, whereas L3373 was a competitive inhibitor. The potency of deethylated and deiodinated compounds (but not of the benzofuran derivatives) for inhibiting T3 binding was twice as high for the beta1-T3R as for the alpha1-T3R. 3) All tested A analogs preferentially interfere with T3 binding to unoccupied receptors. The implications of these findings for the structure-activity relationship are the following: 1) the size of the diethyl-substituted nitrogen group and of the two bulky iodine atoms in the A molecule hamper the binding of A at the T3 binding site of T3Rs; and 2) differences in the hormone-binding domain of alpha1- and beta1-T3Rs are likely to account for the competitive or noncompetitive nature of inhibition of T3 binding by A analogs.

Diurnal variation in rat liver thyroid hormone receptor (TR)-alpha messenger ribonucleic acid (mRNA) is dependent on the biological clock in the suprachiasmatic nucleus, whereas diurnal variation of TR beta 1 mRNA is modified by food intake.

Previous studies have shown a diurnal variation of certain isoforms of thyroid hormone receptors (TR) in rat liver. The genesis of these diurnal changes is still unknown. To clarify whether the biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), is involved, we made selective SCN lesions. Rats with an SCN lesion lost their circadian rhythm of plasma corticosterone and TSH when compared with intact animals. TR alpha 1 and TR alpha 2 mRNA expression of control rats was higher in the light period than in the dark period; changes that were abolished in the rats with SCN lesions. In contrast, liver TR beta 1 mRNA of intact rats showed a diurnal variation that failed to reach statistical significance. To evaluate whether these effects could be explained indirectly by the disappearance of rhythmic feeding behavior in rats with SCN lesions, we performed a second experiment in which otherwise intact animals were subjected to a regular feeding (RF) schedule, with one meal every 4 h. When compared with rats with free access to food, RF only affected TR beta 1 mRNA expression and had no effect on the diurnal changes in TR alpha 1 and TR alpha 2. We conclude that liver TR beta 1 expression is most clearly affected by food intake. Diurnal changes in liver TR alpha 1 and TR alpha 2 are controlled by the biological clock in the SCN but not via changes in the daily rhythm of food intake. The findings may have physiological relevance for diurnal variation of T(3)-dependent gene expression, which is supported by a diurnal variation in the expression of the 5'-deiodinase gene.