Evaluation of body fat composition after linagliptin treatment in a rat model of diet-induced obesity: a magnetic resonance spectroscopy study in comparison with sibutramine.

The effects of linagliptin on fat content in diet-induced obese rats were compared with those of the appetite suppressant sibutramine. Female Wistar rats fed a high-fat diet (HFD) for 3 months received vehicle, linagliptin (10 mg/kg) or sibutramine (5 mg/kg) treatment orally, once daily for 6 additional weeks, while continuing the HFD. Magnetic resonance spectroscopy analysis of fat content was performed at baseline and at the end of the 6-week treatment period. Linagliptin treatment profoundly reduced hepatic fat compared with vehicle, with an effect comparable to that of sibutramine. The vehicle-corrected mean change (95% CI) from baseline in hepatic fat and intramyocellular lipid was -59.0% (-104.3%, -13.6%; p = 0.015) and -62.1% (-131.6%, 7.4%; p = 0.073), respectively, for linagliptin compared with -54.3% (-101.5%, -7.1%; p = 0.027) and -72.4% (-142.4%, -2.4%; p = 0.044), respectively, for sibutramine.

Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: comparison of genomic and proteomic responses and anchoring to histopathological parameters.

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplastic and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.

Proposals for sample size calculation programs.

OBJECTIVES: Numerous sample size calculation programs are available nowadays. They include both commercial products as well as public domain and open source applications. We propose modifications for these programs in order to even better support statistical consultation during the planning stage of a two-armed clinical trial. METHODS: Directional two-sided tests are commonly used for two-armed clinical trials. This may lead to a non-negligible Type III error risk in a severely underpowered study. In the case of a reasonably sized study the question for the so-called auxiliary alternative may evolve. RESULTS: We propose that sample size calculation programs should be able to compute i) Type III errors and the so-called q-values, ii) minimum sample sizes required to keep the q-values below pre-specified levels, and iii) detectable effect sizes of the so-called auxiliary alternatives. CONCLUSIONS: Proposals i and ii are intended to help prevent irresponsibly underpowered clinical trials, whereas the proposal iii is meant as additional assistance for the planning of reasonably sized clinical trials.

Normalization for two-channel microarray data.

OBJECTIVES: In two-channel microarray experiments the measured gene expression levels are affected by many sources of systematic variation. Normalization refers to the process of removing such systematic sources of variation, to make measured intensities within and between slides comparable. Some commonly used normalization methods removing intensity-dependent dye bias and adjusting differences in variability between slides will be reviewed with the main focus on intensity-dependent normalization methods. METHODS: This article describes different intensity-dependent within-slide normalization methods for the log ratios of red and green channel intensities but also refers to single channel normalization methods incorporating all single channels of the slides at once. RESULTS: The described procedures provide a useful approach to remove systematic sources of variation like intensity-dependent dye bias and variability between slides in cDNA microarray experiments. This is illustrated by an experimental data set. CONCLUSIONS: Several reasonable normalization procedures for two-channel microarray data have recently been proposed. Deciding on which method would perform well for a concrete experiment is difficult. Designed spike-in experiments or dilution series with known differences for some selected genes would be helpful to assess the different methods, but may be impractical for most laboratories due to the high costs.

Microarray analysis of newly synthesized RNA in cells and animals.

Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.

Diagnostic performance of spectroscopic and perfusion MRI for distinction of brain tumors.

OBJECTIVE: To assess the value of spectroscopic and perfusion MRI for glioma grading and for distinguishing glioblastomas from metastases and from CNS lymphomas. METHODS: The authors examined 79 consecutive patients with first detection of a brain neoplasm on nonenhanced CT scans and no therapy prior to evaluation. Spectroscopic MRI; arterial spin-labeling MRI for measuring cerebral blood flow (CBF); first-pass dynamic, susceptibility-weighted, contrast-enhanced MRI for measuring cerebral blood volume; and T1-weighted dynamic contrast-enhanced MRI were performed. Receiver operating characteristic analysis was performed, and optimum thresholds for tumor classification and glioma grading were determined. RESULTS: Perfusion MRI had a higher diagnostic performance than spectroscopic MRI. Because of a significantly higher tumor blood flow in glioblastomas compared with CNS lymphomas, a threshold value of 1.2 for CBF provided sensitivity of 97%, specificity of 80%, positive predictive value (PPV) of 94%, and negative predictive value (NPV) of 89%. Because CBF was significantly higher in peritumoral nonenhancing T2-hyperintense regions of glioblastomas compared with metastases, a threshold value of 0.5 for CBF provided sensitivity, specificity, PPV, and NPV of 100%, 71%, 94%, and 100%. Glioblastomas had the highest tumor blood flow values among all other glioma grades. For discrimination of glioblastomas from grade 3 gliomas, sensitivity was 97%, specificity was 50%, PPV was 84%, and NPV was 86% (CBF threshold value of 1.4), and for discrimination of glioblastomas from grade 2 gliomas, sensitivity was 94%, specificity was 78%, PPV was 94%, and NPV was 78% (CBF threshold value of 1.6). CONCLUSION: Perfusion MRI is predictive in distinguishing glioblastomas from metastases, CNS lymphomas and other gliomas vs MRI and magnetic resonance spectroscopy.

Gender-specific expression of liver organic anion transporters in rat.

BACKGROUND: Sex differences in drug pharmacokinetics have been well recognized and gender has been considered a risk factor for adverse events to medications. The aim of this study was to investigate the effect of gender on the expression of hepatocellular transport proteins involved in uptake and secretion of organic anions in rat. MATERIALS AND METHODS: Expression of the rat liver organic anion transporting polypeptides (Oatps) and multidrug resistance proteins (Mrps) was analysed by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis and immunofluorescence microscopy in male and female rats. Regulation of these transport proteins in response to the steroid dehydroepiandrosterone (DHEA) was investigated. RESULTS: In untreated rats, protein expression significantly differed between genders being higher (Mrp2, Mrp3), comparable [Oatp1a1 (Oatp1); Oatp1b2 (Oatp4)] or lower [Oatp1a4 (Oatp2)] in female than in male rat. DHEA treatment over 3 days (100 mg d(-1)) led to a further increase in Mrp3 expression only in female rats. Mrp2 expression was not influenced by DHEA treatment. Oatp1a1 and Oatp1b2 were significantly down-regulated after DHEA treatment in both male and female rats. In contrast, Oatp1a4 was down-regulated in male rats only. CONCLUSIONS: In rat, liver transport proteins of the Oatp and Mrp family are expressed and regulated in a gender-specific manner according to sexual differences in the hepatic metabolism of steroids and drugs. These findings may partly explain the well-known sex differences in hepatic handling of organic anions.