Metabolic Profiling of Multiorgan Samples - ACS Publications

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Metabolic profiling of multi-organ samples - Evaluation of MODY5/RCAD mutant mice Frida Torell, Kate Bennett, Silvia Cereghini, Mélanie Fabre, Stefan Rännar, Katrin Lundstedt-Enkel, Thomas Moritz, Cécile Haumaitre, Johan Trygg, and Torbjörn Lundstedt J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00821 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Journal of Proteome Research

Metabolic profiling of multi-organ samples - Evaluation of MODY5/RCAD mutant mice. Frida Torell1,2, Kate Bennett3, Silvia Cereghini4,5,6, Mélanie Fabre4,5,6, Stefan Rännar3, Katrin Lundstedt-Enkel3,7, Thomas Moritz3,8, Cécile Haumaitre4,5,6, Johan Trygg1,+,*, and Torbjörn Lundstedt3,9,+ 1 Computational Life Science Cluster (CLiC), Department of Chemistry, Umeå University, 90187, Sweden 2 Accelerator Lab (ACL), Karlsruhe Institute of Technology, 76344, Germany 3 AcureOmics AB, Umeå, 90736, Sweden 4 CNRS, UMR7622, 75005, Paris, France 5 Sorbonne Universites, UPMC, UMR7622, 75005, Paris, France 6 Inserm U-1156, Paris, France 7 Department of Organismal Biology, Uppsala University, 75236, Sweden 8 Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 87 Umeå, Sweden 9 Department of Organic Pharmaceutical Chemistry, Uppsala University, 75123, Sweden + these authors contributed equally to this work *e-mail: [email protected]

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Phone: +46907866917. ORCID: Johan Trygg 0000-0003-3799-6094 ABSTRACT In the present study, we performed a metabolomics analysis to evaluate a MODY5/RCAD mouse mutant line as a potential model for HNF1B-associated diseases. Gas chromatography time-offlight mass spectrometry (GC-TOF-MS) of gut, kidney, liver, muscle, pancreas and plasma samples uncovered the tissue specific metabolite distribution. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) was used to identify the differences between MODY5/RCAD and wild-type mice, in each of the tissues. The differences included e.g. increased levels of amino acids in the kidneys and reduced levels of fatty acids in the muscles of the MODY5/RCAD mice. Interestingly, campesterol was found in higher concentrations in the MODY5/RCAD mice, with a 4-fold and 3-fold increase in kidneys and pancreas, respectively. As expected, the MODY5/RCAD mice displayed signs of impaired renal function in addition to disturbed liver lipid metabolism, with increased lipid and fatty acid accumulation in the liver. From a metabolomics perspective, the MODY5/RCAD model was proven to display a metabolic pattern similar to what would be suspected in HNF1B-associated diseases. These findings were in line with the presumed outcome of the mutation, based on the different anatomy and function of the tissues, as well as the effect of the mutation on development. KEY WORDS: HNF1B-associated diseases, metabolomics, OPLS-DA, multi-organ samples, MODY5, RCAD, mouse model

INTRODUCTION 2 ACS Paragon Plus Environment

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Maturity onset diabetes of the young (MODY) is a clinically heterogeneous group of monogenic disorders caused by single gene mutations (e.g. HNF4A, GCK, HNF1A, HNF1B, etc.) characterized by autosomal dominant inheritance of early-onset, non-insulin-dependent diabetes.1, 2 Fajans and Conn were the first to use the expression “maturity-onset type diabetes of childhood or of the young”, in 1964 at the Fifth Congress of the International Diabetes Federation in Toronto.3,

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Since then, approximately thirteen types of MODY have been

identified and characterized.5 The minimum prevalence of MODY is approximately 100 per million population.6 Diagnosis is based on genetic testing. However, the varying referral rate to genetic tests results in over 80% of cases being undiagnosed. MODY patients are often misdiagnosed with either Type 1 Diabetes or Type 2 Diabetes.4, 6 This leads to the administration of treatments not optimal for their condition. MODY5, also referred to as Renal Cysts And Diabetes Syndrome (RCAD), results from mutations in the Hepatocyte Nuclear Factor 1 homeobox B (HNF1B) gene, also known as Transcription Factor 2 (TCF2). The onset age of MODY5 varies.7, 8 The prevalence of MODY5 among all clinically diagnosed MODY is reported to be 2–6%.4, characterized using genomic approaches.10,

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MODY5/RCAD has been

More than 100 different mutations have been

reported, comprising base substitutions, small insertion–deletions and whole-gene deletions. Antenatal discovery, family history, and organ involvement can be used to identify patients who should undergo HNF1B gene analysis.12 However, no genetic-phenotype correlation could be identified. The disease is manifested during gestation. Indeed, both pancreatic agenesis or hypoplasia, genital tract abnormalities and multicystic renal dysplasia have been observed in fetuses carrying different HNF1B mutations.13-15



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HNF1B-associated disease is a multi-system disorder. A whole spectrum of associated phenotypes have been reported.16-18 These include renal cysts and renal hypoplasia19, early onset diabetes19, pancreatic hypoplasia and pancreatic exocrine dysfunction16, malformations21, liver and intestinal

abnormalities22,

23

20

, genital tract

, autism spectrum disorders,

hypomagnesaemia, hyperuricaemia and gout. MODY5/RCAD patients display a wide variability in the severity as well as clinical symptomology, even within the same family. This autosomal dominant disorder is associated with a spectrum of phenotypes where renal abnormalities are most common followed by pancreatic and genital tract abnormalities.18 These multi-organ phenotypes are consistent with the reported critical roles of HNF1B in the development of several mouse organs in particular the kidneys, liver and pancreas, from the early stages of embryogenesis.24-30 In previous metabolomic studies investigating different MODY subtypes, serum samples from MODY 1, 2 and 331 as well as urine samples from MODY 2 and 332 have been analyzed. Low levels of free fatty acids and triglycerides were found to be associated with the metabolic serum profile of MODY 2 patients, which is a non-progressive MODY type.31 The metabolic profile of urine from patients with MODY 3 were found to be associated with high levels of glucose.32 To the authors knowledge no metabolomic study has been reported in MODY5/RCAD patients, or previous Hnf1b genetically modified mice. The concentration of metabolites in biofluids reflects systemic effects of simultaneously occurring processes in different tissues and cell types in an organism. Hence they provide a snapshot of metabolic activity in the organism at a given time point. The interest for analyzing tissue samples and biopsies has increased since they can add insight to the origin of the metabolic differences observed in these biofluids. 4 ACS Paragon Plus Environment

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In the present study, we performed a metabolomic study in a MODY5/RCAD mouse mutant line, generated by the introduction of a previously identified human point mutation in the locus of Hnf1b. This mouse line was generated since the homozygous deletion of Hnf1b (Hnf1b-/-) in mice is embryonically lethal33 whereas the heterozygous Hnf1b-deficient mice (Hnf1b+/-), unlike humans, show no phenotype33,

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. This mouse line, thereafter referred to as MODY5/RCAD

mutants, exhibited several features of the human disease. In particular, it presents renal abnormalities, with cystic kidneys and slow progressive renal dysfunctions, pancreatic dysfunction with glucose intolerance and genital tract malformations. Whereas the liver phenotype remains to be examined (S. Cereghini, C. Haumaitre; manuscripts in preparation). Therefore this mouse model is potentially useful for the analysis of the disease. Applying a metabolomics approach to both plasma and tissue samples allows the metabolic status of the different organs and plasma to be identified. As MODY5/RCAD affects multiple organs, metabolomics from several tissues normally expressing Hnf1b (e.g. kidney, pancreas, liver)18 could clearly be important in the context of the analysis of this disease. By utilizing metabolomics in this way, the location of the metabolic differences is revealed and information is provided regarding the level of organ contribution to what is observed in plasma. To further evaluate the MODY5/RCAD mouse line as a potential model for HNF1B-associated diseases, a total of six sample types (gut, kidney, liver, muscle, pancreas and plasma) were harvested from eight-month-old male mice and analyzed using a metabolomic approach utilizing GC-TOF MS.

EXPERIMENTAL PROCEDURES



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Samples Eight-month-old wild-type male mice of a mixed background 129sv x C57Bl/6N (n=4) and heterozygous male mice carrying a human point mutation in the locus of Hnf1b (n=4) were individually placed in metabolic cages (Metabolic 5/13 cage for mice, TecniplastTM, UK), with access to food (ref A04-10 in powder, SAFE (Scientific animal food & engineering), France) and water. The animals were kept 5 days in the metabolic cages, 2 days as an adaptation period and 3 days of experiment. On each experimental day, animals were weighed. Food and water intake, urine volume and fecal weight for each mouse were recorded. After 4h fasting, 50µl of blood from the tail was recovered in heparin tubes (Microvette CB300 LH, Sarstedt, Germany). Plasma was obtained from the blood sample by centrifugation (2000 rpm, 5 min at 20°C), and frozen at 80°C. On the 5th day of the experiment, animals were anesthetized with a ketamine and xylazine solution by intraperitoneal injection, a blood sample was obtained by retro-orbital bleeding performed with a heparinized Pasteur pipette, and plasma was prepared as described above. Animals were killed by cervical dislocation. Pancreas, liver, gut, kidney and muscle tissues were removed and dissected in cold HBSS solution (Hanks balanced salt solution, Life Technologies, US). Samples of each organ were washed in HBSS, collected in Cryo tubes, frozen in liquid nitrogen, and stored at -80°C. Animal experiments and the experimental protocols were approved by, and conducted in accordance with French and European ethical legal guidelines and the local ethical committee for animal care (Comité déthique en expérimentation animale Charles Darwin N°5, approval numbers N° 01508.01 and N° 04817.02). Materials All standard reagents were of analytical grade or equivalent and obtained from Sigma-Aldrich (St Louis, MO, USA), Merck (Darmstadt, Germany) and J.T. Baker (Phillipsburg, NJ, USA). N6 ACS Paragon Plus Environment

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Methyl-N-trimethylsilyltrifluoro-acetamide (MSTFA) plus 1% trimethylchlorosilane (TMCS) and pyridine were obtained from Thermo Fisher Scientific (Rockford, IL, USA). Only Milli-Q water was used. The stable isotope-labelled internal standards [13C5]-proline, [2H4]-succinic acid, [13C5,

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N]-glutamic acid, [1,2,3-13C3]-myristic acid, [2H7]-cholesterol and [13C4]-disodium α-

ketoglutarate were purchased from Cambridge Isotope Laboratories (Andover, MA); [13C6]glucose, [13C12]-sucrose, [13C4]-hexadecanoic acid and [2H4]-putrescine were purchased from Campro (Veenendaal, The Netherlands) and [2H6]-salicylic acid was obtained from Icon (Summit, NJ). Stock solutions of each internal standard were prepared in either Milli-Q water or methanol to a concentration of 500 ng/µL. The internal standards were selected on the basis that they represented several classes of metabolites and eluted at different times spanning the whole GC-chromatogram.35, 36 Metabolite extraction from tissues The samples hence forth referred to as gut consisted mainly of the duodenum. The kidney, liver, and pancreas samples refer to the homogenized whole organs. The muscle samples were skeletal muscle tissue from the hindlimb. Samples were ground to a fine powder under liquid nitrogen. A total of 1 ml methanol-chloroform-water (3:1:1), containing all eleven isotopically labelled internal standards (7 ng/µl each) was added to 10 mg of frozen tissue. Samples were extracted using a MM 400 Vibration Mill (Retsch GmbH & Co. KG, Haan, Germany) at a frequency of 30 Hz for 3 min. A 3 mm tungsten carbide bead (Retsch GmbH & Co. KG, Haan, Germany) was added to each tube prior to mixing to increase the extraction efficiency. The beads were removed and the samples were centrifuged at 18 620 g for 15 min at 4°C. A volume of 200 µl supernatant was transferred to a GC vial and evaporated to dryness in a SpeedVac concentrator (Savant Instrument, Framing-dale, NY, USA). All samples were stored at -80°C until analysis. 7 ACS Paragon Plus Environment


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Metabolite extraction from plasma Frozen plasma was thawed at room temperature for 10 min and stored on ice (4°C). Plasma samples were prepared for GC-TOF-MS analysis by adding 900 µl extraction mix (methanol:water, 8:2, v/v), containing all eleven isotopically labelled internal standards (7 ng/µl), to 100 µl aliquots of plasma. Each sample was extracted vigorously using a MM 400 Vibration Mill (Retsch GmbH & Co. KG, Haan, Germany) at a frequency of 30 Hz for 3 min, followed by centrifugation at 18 620 g for 15 min at 4°C. A volume of 200 µl supernatant was transferred to a GC vial and evaporated to dryness in a SpeedVac concentrator (Savant Instrument, Framingdale, NY, USA). Derivatisation of samples A 30 µl sample of methoxyamine (15 µg/µl) in pyridine was added to each GC vial and the resultant mixture was shaken vigorously for 10 min. Plasma samples were also incubated for 1 h in an oven at 70°C. Methoxymation was performed at room temperature for 16 h, followed by the addition of 30 µl MSTFA with 1% TMCS to each sample (brief vortex, 10 s). Samples were left at room temperature for 1 h to allow silylation to occur, followed by the addition of 30 µl heptane (containing 15 ng/µl methyl stearate as an internal standard) and a brief vortex for 10 s. For selected samples technical replicates were also performed, where the whole extraction process was repeated (in triplicate) on the same plasma, liver and kidney sample. GC-TOF-MS analysis The samples were randomized throughout the chemical analysis. Methyl stearate standard was analyzed every 10 samples in order to monitor the sensitivity of the instrument. A volume of 1 µl of each derivatised sample was injected splitless by a CTC Combi Pal autosampler (CTC 8 ACS Paragon Plus Environment

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Analytics AG, Zwingen, Switzerland) into an Agilent 6980 GC equipped with a 10 m x 0.18 mm i.d. fused-silica capillary column chemically bonded with 0.18-µm DB 5-MS stationary phase (J&W Scientific Folsom, CA). The injector temperature was set to 270°C. Helium was used as the carrier gas at a constant flow rate of 1 mL min−1 through the column. For every analysis, the purge time was set to 60 s at a purge flow rate of 20 mL min−1 and an equilibrium time of 1 min. The column temperature was held initially at 70°C for 2 min, then increased to 320°C at a rate of 30°C min−1, where it was held for 2 min. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer (Leco Corp., St Joseph, MI). The ion source and transfer line temperatures were set to 200°C and 250°C, respectively. Ions were generated by a 70-ev electron beam at a current of 2.0 mA. Masses were acquired in the mass range 50-800 m/z at a rate of 30 spectra s−1. The acceleration voltage was turned on after a solvent delay of 150 s. The detector voltage was 1670 V. Data processing and mass spectra identification Nonprocessed MS files from GC/TOFMS analysis were exported in NetCDF format to MATLAB software 7.11 (Mathworks, Natick, MA), where all data pretreatment procedures, such as baseline correction, chromatogram alignment, time-window setting and multivariate curve resolution (MCR)37 were performed using custom scripts. Automatic peak detection and mass spectrum deconvolution with ChromaTof software were performed using a peak width set to 2 s. For the identification of metabolites, NIST MS Search 2.0 software was used to compare the mass spectra of all detected compounds with spectra in the NIST library 2.0. In addition, the in-house mass spectra library established by Umea Plant Science Centre and the mass spectra library

maintained

by

the

Max

Planck

Institute

in

Golm

(http://csbdb.mpimp-

golm.mpg.de/csbdb/gmd/gmd.html) were used. This library has been created on site and contains 9 ACS Paragon Plus Environment


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both retention index and mass spectra information. Annotations were based on both mass spectra and retention index. The mass spectra were also validated manually, i.e. manual comparison between obtained mass spectrum and library mass spectrum. The retention index comparison was performed, with a retention index deviation

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