S.G. Moesgaard

Characterisation of Gut Microbiota in Ossabaw and Göttingen Minipigs as Models of Obesity and Metabolic Syndrome

Background Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs. Methods and Findings The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs’ cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine. Conclusion Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.

Effects of high-fat feeding and fasting on ghrelin expression in the mouse stomach.

Ghrelin is a peptide identified as an endogenous ligand for the growth hormone secretagogue receptor. Studies have shown that ghrelin stimulates growth hormone, promotes food intake and decreases energy expenditure. Furthermore, feeding status seems to influence plasma ghrelin levels, as these are increased during fasting, whereas feeding and oral glucose intake reduce plasma ghrelin. This study examined whether standardized obesity and fasting affect cellular expression of ghrelin. Specimens from the gastrointestinal tract of fed or 18-h fasted, low-fat or high-fat fed (10 weeks on diet) C57BL/6J mice were studied by immunocytochemistry (ICC) for ghrelin and in situ hybridization (ISH) for ghrelin mRNA. Ghrelin was expressed in especially the corpus but also the antrum of the stomach of all groups studied. Cells positive for ghrelin and ghrelin mRNA in the stomach were reduced in high-fat fed mice. In contrast, ghrelin expression was not affected by fasting. The reduction in ghrelin expression in the high-fat fed mice was associated with a reduction in plasma levels of ghrelin, whereas after fasting, when expression rate was not altered, there was an increase in plasma ghrelin. In conclusion, ghrelin is highly expressed in the corpus and antrum of the stomach of C57BL/6J mice. This expression is reduced in obesity, whereas fasting has no effect.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Left Ventricular Twist and Circumferential Strain in Dogs with Myxomatous Mitral Valve Disease

BACKGROUND During the cardiac cycle, the ventricle undergoes a twisting motion because of the oblique orientation of the left ventricular (LV) myofibers. This can be quantified by speckle-tracking echocardiography (STE). In mitral regurgitation (MR) in humans, the short axis deformation has been suggested as being pivotal to LV function. Decreased and delayed LV twist has been described in experimental MR, but has not been studied in myxomatous mitral valve disease (MMVD). HYPOTHESES (1) Magnitude (CSt) and rate (CSRs) of systolic circumferential deformation decrease before the onset of congestive heart failure (CHF); (2) magnitude and rate of LV twist decrease, and onset of untwist is delayed, with increasing MMVD severity. ANIMALS A total of 97 privately owned small- to medium-sized dogs. METHODS Severity of MMVD was assessed by echocardiography and presence of clinical signs of CHF. Magnitude and rate of LV twist and circumferential deformation were evaluated by STE. RESULTS Dogs with CHF receiving treatment had increased CSt, CSRs, early diastolic untwisting rate, and delayed onset of untwist compared to dogs with minimal MMVD and increased systolic twist compared to dogs with mild MMVD (all P < .01). CSt and time to onset of untwist increased with echocardiographic variables of MR severity (all P < .002). CSRs and several LV twist variables decreased with increasing systolic LV internal diameter (all P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE No STE-derived variable was decreased before onset of CHF. In dogs with CHF receiving treatment, the delayed onset of relaxation might indicate LV dysfunction and the hyperdynamic CSt and LV twist reflect compensatory mechanisms.

Holter Monitoring of Small Breed Dogs with Advanced Myxomatous Mitral Valve Disease with and without a History of Syncope

Background Syncope is a transient loss of consciousness occasionally occurring in dogs with advanced myxomatous mitral valve disease (MMVD). Objective (1) To study ECG changes during syncopal episodes in dogs with advanced MMVD and (2) to compare the occurrence of arrhythmias and changes in heart rate variability (HRV) between dogs with advanced MMVD with and without a history of syncope. Animals Forty‐three privately owned dogs (<15 kg) with advanced MMVD: 21 with and 22 without a history of syncope. Methods Prospective study with dogs recruited for an evaluation including history, physical examination, echocardiography, and arrhythmia and HRV analysis performed on 24‐hour Holter recordings. Results A syncopal episode was observed during Holter monitoring in 4 dogs: 3 dogs had sinus rhythm and 1 dog had sinus arrest followed by escape rhythm. An arrhythmia variable representing sinus arrhythmia was significantly lower in dogs with a history of syncope than in those without (P = .008). Eight of 26 HRV variables were significantly different between dogs with and without a history of syncope. Conclusions and Clinical Importance Compared with dogs without a history of syncope, dogs with advanced MMVD and a history of syncope did not have a higher occurrence of arrhythmias, but had less sinus arrhythmia, and had changes in HRV variables representing decreased overall HRV, decreased parasympathetic, and increased sympathetic modulation of heart rate.

Serotonin markers show altered transcription levels in an experimental pig model of mitral regurgitation.

Serotonin (5-hydroxytryptamine, 5-HT) signalling is implicated in the pathogenesis of myxomatous mitral valve disease (MMVD) through 5-HT1B receptor (R), 5-HT2AR and 5-HT2BR-induced myxomatous pathology. Based on increased tryptophan hydroxylase-1 (TPH-1) and decreased serotonin re-uptake transporter (SERT) in MMVD-affected valves, increased valvular 5-HT synthesis and decreased clearance have been suggested. It remains unknown how haemodynamic changes associated with mitral regurgitation (MR) affect 5-HT markers in the mitral valve, myocardium and circulation. Twenty-eight pigs underwent surgically induced MR or sham-operation, resulting in three MR groups: control (CON, n = 12), mild MR (mMR, n = 10) and severe MR (sMR, n = 6). The gene expression levels of 5-HT1BR, 5-HT2AR, 5-HT2BR, SERT and TPH-1 were analysed using quantitative PCR (qPCR) in the mitral valve (MV), anterior papillary muscle (AP) and left ventricle (LV). MV 5-HT2BR was also analysed with immunohistochemistry (IHC) in relation to histological lesions and valvular myofibroblasts. All 5-HTR mRNAs were up-regulated in MV compared to AP and LV (P <0.01). In contrast, SERT and TPH-1 were up-regulated in AP and LV compared to MV (P <0.05). In MV, mRNA levels were increased for 5-HT2BR (P = 0.02) and decreased for SERT (P = 0.03) in sMR vs. CON. There were no group differences in 5-HT2BR staining (IHC) but co-localisation was found with α-SMA-positive cells in 91% of all valves and with 33% of histological lesions. In LV, 5-HT1BR mRNA levels were increased in sMR vs. CON (P = 0.01). In conclusion, these data suggest that MR may affect mRNA expression of valvular 5-HT2BR and SERT, and left ventricular 5-HT1BR in some pigs.

Alpha-smooth muscle actin and serotonin receptors 2A and 2B in dogs with myxomatous mitral valve disease.

Canine Myxomatous mitral valve disease (MMVD) is an age-related disease. Serotonin (5-HT) is implicated in the pathogenesis as locally-produced or platelet-derived. Involvement of the 5-HT2A receptor (R) and 5-HT2BR in the induction of myxomatous-mediating valvular myofibroblasts (MF) has been suggested. In an age-matched population of dogs with non-clinical and clinical MMVD, the objectives were to investigate (1) gene expression of 5-HT2AR and 5-HT2BR, (2) protein expression and spatial relationship of 5-HT2AR, 5-HT2BR and MF in the mitral valve (MV) and the cardiac anterior papillary muscle (AP) and (3) serum 5-HT concentrations. Gene expression of 5-HT2BR was significantly higher in MV and AP among dogs with clinical MMVD. This was not found for 5-HT2BR protein expression, though association of 5-HT2BR with myxomatous pathology and co-localization of 5-HT2BR and MF in MV and AP support a functional relationship, perhaps perpetuation of clinical MMVD. 5-HT2AR-expression and serum 5-HT showed no differences between groups.

Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and transforming growth factor-β (TGF-β) in advanced canine myxomatous mitral valve disease.

This study investigated mitral valve and myocardial protein and gene expression of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and transforming growth factor-β (TGF-β) and plasma MMP and TGF-β concentrations in age-matched dog groups euthanized due to either advanced myxomatous mitral valve disease (MMVD) or other reasons. Furthermore, echocardiographic data and lumen/area ratio were correlated with tissue and plasma levels of MMPs, TIMPs and TGF-βs. Mitral valve and myocardial gene expression of MMP2, MMP14, TGF-β1 and TGF-β2 were increased and plasma MMP9 was decreased in advanced MMVD dogs. Myocardial gene expression of TIMP2 and TIMP3 were increased in advanced MMVD. All affected markers correlated to echocardiographic parameters. Significantly narrowed lumen/area ratio was associated with increased myocardial expression of MMP2, MMP14, TIMP2 and TIMP3. No differences in tissue protein expression were recorded. MMP2, MMP14, TIMP2, TIMP3, TGF-β1 and TGF-β2 appear to play a local role in the development of advanced MMVD.

Functional network analysis of obese and lean Göttingen minipigs elucidates changes in oxidative and inflammatory networks in obese pigs.

The Göttingen minipig model of obesity is used in pre-clinical research to predict clinical outcome of new treatments for metabolic diseases. However, treatment effects often remain unnoticed when using single parameter statistical comparisons due to the small numbers of animals giving rise to large variation and insufficient statistical power. The purpose of this study was to perform a correlation matrix analysis of multiple multi-scale parameters describing co-segregation of traits in order to identify differences between lean and obese minipigs. More than 40 parameters, ranging from physical, cardiovascular, inflammatory and metabolic markers were measured in lean and obese animals. Correlation matrix analysis was performed using permutation test and bootstrapping at different levels of significance. Single parameter comparisons yielded significant differences between lean and obese animals mainly for known physical traits. On the other hand, functional network analysis revealed new co-segregations, particularly in the domain of inflammatory and oxidative stress markers in the obese animals that were not present in the lean. Functional networks of lean or obese minipigs could be utilised to assess drug effects and predict changes in parameters with a certain degree of precision, on the basis of the networks confidence intervals. Comparison of functional networks in minipigs with those of human clinical data may be used to identify common parameters or co-segregations related to obesity between animal models and man.

Strong association between activated valvular interstitial cells and histopathological lesions in porcine model of induced mitral regurgitation

Strong association between activated valvular interstitial cells and histopathological lesions in porcine model of induced mitral regurgitation☆ S.E. Cremer , N.E. Zois , S.G. Moesgaard , N. Ravn , S. Cirera , J.L. Honge , M.H. Smerup , J.M. Hasenkam , E. Sloth , P.S. Leifsson , T. Falk , M.A. Oyama , C. Orton , T. Martinussen , L.H. Olsen a,⁎,1 a Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark b Department of Clinical Biochemistry, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark c Novo Nordisk A/S, Maaloev, Denmark d Department of Cardiothoracic and Vascular Surgery, Aarhus University Hospital, Aarhus, Denmark e Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark f Department of Anaesthesiology and Intensive Care Medicine, Aarhus University Hospital, Aarhus, Denmark g Department of Clinical Studies, University of Pennsylvania, Philadelphia, PA, USA h Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA i Department of Biostatistics, University of Copenhagen, Copenhagen, Denmark