Quantifying energy expenditure in Göttingen Minipigs with the 13C-bicarbonate method under basal and drug-treated conditions.

Effective treatments of obesity focusing on energy expenditure (EE) are needed. To evaluate future EE-modulating drug candidates, appropriate animal models and methods to assess EE are needed. This study aimed to evaluate the stable isotope 13C-bicarbonate method (13C-BM) for estimating EE in Göttingen minipigs under basal and drug-treated conditions. Four experiments (Expt.1-4) were conducted to assess: 1) the 13C-BM reproducibility using breath sample collection (n = 8), on two consecutive days, 2) the effect of two dose levels (5 and 10 mg/kg body weight (BW)) of the mitochondrial uncoupler dinitrophenol (DNP) in a crossover design (n = 8), 3) sampling method agreement; blood vs. exhaled air (n = 6) and 4) 13C-BM using constant isotope infusion compared with indirect calorimetry (IC) (n = 3). Results correlated significantly (p < 0.001) between days (Expt.1), with an average coefficient of variance of 5.4 ± 2.3%. Administration of 10 mg DNP/kg BW increased (p < 0.01) EE by 33.2 ± 6.4% (Expt.2). Results based on different sampling methods correlated significantly (p < 0.001) and EE increased after 10 mg DNP/kg BW (p < 0.05) in Expt.3. However, results based on blood sampling were significantly higher (p < 0.01) than those of exhaled air. No effect of DNP and significantly different EE results (p < 0.05) was observed in a limited number of animals, when constant isotope infusion and blood sampling was compared with IC (Expt.4). In conclusion, the 13C-BM is useful for investigating treatment effects on EE in minipigs. However, further validation under standardized conditions is needed to provide accurate estimates of the 13C recovery factor and respiratory quotient, both of decisive importance when using the 13C-BM.

Influence of general anaesthesia on circulating biomarkers of glucose metabolism in pigs.

Pigs are widely used in metabolic research with procedures often requiring general anaesthesia. The aim was to investigate the effect of four different anaesthetic protocols: 1) isoflurane inhalation, 2) propofol infusion, 3) a mixture of tiletamine, zolazepam, medetomidine, ketamine and butorphanol (TZMKB)) and 4) ketamine combined with midazolam and xylazine (KMX)) on selected biomarkers during basal and glucose stimulated conditions. Eight domestic pigs were included in a cross-over design. Plasma concentrations of glucose, insulin, C-peptide, glucagon, cortisol, triglycerides, total cholesterol, aspartate amino transferase and alanine amino transferase, creatinine, urea, fructosamine, albumin, free fatty acids (FFAs) and glycerol were measured at baseline, during 2 h of anaesthesia and during 1 h of recovery. Intravenous glucose tolerance test (IVGTT, 0.5 g glucose/kg) was performed after 1 h of anaesthesia. Glucose disappearance rate and areas under the insulin, C-peptide and glucagon curves from the IVGTT were calculated. All four anaesthetic protocols affected glucose metabolism parameters significantly compared with un-anaesthetised pigs, which was particularly evident during IVGTT and for TZMKB and KMX anaesthesia. Propofol additionally influenced the plasma concentrations of triglycerides, FFAs and glycerol significantly. The remaining circulating biomarkers were largely unaffected by anaesthesia. These data underline the importance of considering the anaesthetic protocol in porcine studies of circulating metabolic biomarkers.

Marked gut microbiota dysbiosis and increased imidazole propionate are associated with a NASH Göttingen Minipig model.

BACKGROUND: Gut microbiota dysbiosis is associated with the development of non-alcoholic steatohepatitis (NASH) through modulation of gut barrier, inflammation, lipid metabolism, bile acid signaling and short-chain fatty acid production. The aim of this study was to describe the impact of a choline-deficient amino acid defined high fat diet (CDAHFD) on the gut microbiota in a male Göttingen Minipig model and on selected pathways implicated in the development of NASH. RESULTS: Eight weeks of CDAHFD resulted in a significantly altered colon microbiota mainly driven by the bacterial families Lachnospiraceae and Enterobacteriaceae, being decreased and increased in relative abundance, respectively. Metabolomics analysis revealed that CDAHFD decreased colon content of short-chain fatty acid and increased colonic pH. In addition, serum levels of the microbially produced metabolite imidazole propionate were significantly elevated as a consequence of CDAHFD feeding. Hepatic gene expression analysis showed upregulation of mechanistic target of rapamycin (mTOR) and Ras Homolog, MTORC1 binding in addition to downregulation of insulin receptor substrate 1, insulin receptor substrate 2 and the glucagon receptor in CDAHFD fed minipigs. Further, the consequences of CDAHFD feeding were associated with increased levels of circulating cholesterol, bile acids, and glucagon but not total amino acids. CONCLUSIONS: Our results indicate imidazole propionate as a new potentially relevant factor in relation to NASH and discuss the possible implication of gut microbiota dysbiosis in the development of NASH. In addition, the study emphasizes the need for considering the gut microbiota and its products when developing translational animal models for NASH.

NASH-inducing Diets in Göttingen Minipigs.

BACKGROUND: Owing to the human-like physiology, a minipig model of nonalcoholic steatohepatitis (NASH) could be valuable. Pigs, however, rarely develop substantial hepatic steatosis, even when fed diets with high fat, fructose, and cholesterol (FFC) content. The potential of choline-deficient, amino acid-defined high-fat diets (CDAHFD) was therefore evaluated in Göttingen Minipigs. METHODS: Castrated male Göttingen Minipigs were fed either chow (n = 5) or one of the three NASH diets: FFC (n = 5), CDAHFD with sucrose (CDAHFD-S; n = 4), or fructose (CDAHFD-F; n = 4) for 8 weeks. Liver and blood samples were collected after 2 weeks and at termination. RESULTS: Compared with chow, the body weight was higher after FFC (9.8 ± 0.4 versus 8.5 ± 1.2 kg; mean ± SD) and less after CDAHFD-S (6.4 ± 0.8 kg) and CDAHFD-F (6.9 ± 0.8 kg). Liver weight per kg body weight was significantly increased in all 3 NASH groups (FFC 2.1 times; and both CDAHFD diets 3.1 times). Histologically, pronounced macrovesicular steatosis developed only in the CDAHFD groups. Inflammation was present in all three NASH groups. In the CDAHFD groups, inflammatory cells formed crown-like structures around steatotic hepatocytes. Sirius red staining revealed mild fibrosis in the two CDAHFD groups with the fibrotic potential being further supported by immunohistochemical staining for activated stellate cells and gene expression analyses. No noticeable differences were found between CDAHFD-S and CDAHFD-F. CONCLUSIONS: Göttingen Minipigs fed CDAHFD developed pronounced steatosis with inflammation around steatotic hepatocytes and incipient fibrosis, thereby showing potential as a model for human NASH. Further studies are needed to investigate the period needed for marked fibrosis to develop.

Long-acting CCK analogue NN9056 lowers food intake and body weight in obese Göttingen Minipigs.

BACKGROUND/OBJECTIVES: Cholecystokinin (CCK) is a regulator of appetite and energy intake in man. The aim of this study was to determine the effect of NN9056, a long-acting CCK-1 receptor-selective CCK analogue, on food intake and body weight (BW) in obese Göttingen Minipigs. SUBJECTS/METHODS: Tolerability of NN9056 and acute effects on food intake, pancreas histology, amylase and lipase levels were assessed in lean domestic pigs in doses up to 100 nmol/kg (n = 3-4). Subsequently, obese Göttingen Minipigs were treated subcutaneously (s.c.) once daily for 13 weeks with vehicle, NN9056 low dose (regulated from 5 to 2 nmol/kg) or NN9056 high dose (10 nmol/kg) (n = 7-8). Food intake was measured daily and BW twice weekly. At the end of the treatment period, an intravenous glucose tolerance test (IVGTT) and a 24-h exposure profile was obtained. Data are mean ± SD. RESULTS: The acute studies in domestic pigs showed significant and dose-dependent effect of NN9056 on food intake, acceptable tolerability and no histopathological signs of pancreatitis. Sub-chronic treatment in obese Göttingen Minipigs was also well tolerated and accumulated food intake was significantly lower in both treated groups compared to vehicle, with no significant difference between the dose levels of NN9056 (41.8 ± 12.6, 51.5 ± 13.8 and 86.5 ± 19.5 kg in high-dose, low-dose and vehicle groups, respectively, p = 0.012 and p < 0.0001 for low and high dose vs. vehicle, respectively). Accordingly, there was a weight loss in both treated groups vs. a weight gain in the vehicle group (-7.2 ± 4.6%, -2.3 ± 3.2% and 12.3 ± 3.9% in the high-dose, low-dose and vehicle groups, respectively, p < 0.0001 for both vs. vehicle). IVGTT data were not significantly different between groups. CONCLUSION: NN9056, a long-acting CCK-1 receptor-selective CCK analogue, significantly reduced food intake and BW in obese Göttingen Minipigs after once daily s.c. dosing for 13 weeks.

Lipid Embolism in Obese Göttingen Minipigs.

Pigs are used as a model of human obesity, both for metabolic characterization and for evaluation of pharmacological interventions. Over a period of 7 years, acute death or clinical signs requiring immediate euthanasia were observed in 12 obese Göttingen minipigs (GMs) included in different pharmacological studies. The GM were fed ad libitum on normal chow-diet and the unscheduled deaths occurred in animals treated with drug candidates as well as in untreated animals. The most prominent clinical signs requiring euthanasia included varying degrees of respiratory distress; and on histopathological examination, thickening of the alveolar septa due to vacuolation was observed throughout the lung in 10 of the 12 animals. Furthermore, vacuolation in glomeruli of the kidney was detected in 9 of the 10 animals. Oil red O staining of cryosections demonstrated that the vacuoles both in lung and kidney contained lipid, and immunohistochemistry with anti-von Willebrand factor and transmission electron microscopy revealed that the lipid was localized in the lumen of blood vessels establishing the occurrence of fatal pulmonary lipid embolism. Additionally, lipogranulomatous inflammation in the abdominal adipose tissue was observed in all the GMs with lipid emboli.

Towards a Göttingen minipig model of adult onset growth hormone deficiency: evaluation of stereotactic electrocoagulation method.

BACKGROUND: Adult onset growth hormone (GH) deficiency (AGDH) is a potentially underdiagnosed condition, caused by damage to the pituitary gland. AGHD is treated with growth hormone replacement therapy. A large variety of clinical symptoms and changes in the metabolic homeostasis can be observed and quantified. New large animal models are needed for future drug development. NEW METHOD: In this study, we evaluate methods for a new large non-primate animal model of GH deficiency in post pubertal Göttingen Minipigs (minipig). Lesions in the pituitary gland were made by stereotaxic monopolar thermo-coagulation guided by magnetic resonance imaging (MRI), and pituitary function was evaluated using insulin tolerance test (ITT) with measurements of growth hormone secretion induced by hypoglycemia. RESULTS: Lesions were successfully applied to the pituitary gland without any damage to surrounding tissue including the hypothalamus, which was confirmed by post-operative MRI and post mortem histology. Plasma levels of GH during ITT showed no decrease in secreted levels one week after surgery compared to levels obtained before surgery. COMPARISON WITH EXISTING METHODS: Compared to other GH insufficiency models, eloquent brain tissue is spared. Furthermore, alternatively to rodent models, a large animal model would allow the use of human intended equipment to evaluate disease. Using the minipig avoids social, economical and ethical issues, compared with primates. CONCLUSION: The lesions did not remove all GH production, but proof of concept is demonstrated. In addition, the ITT is presented as a safe and efficient method to diagnose GH deficiency in minipigs.

18F-FDG PET/MR-imaging in a Göttingen Minipig model of atherosclerosis: Correlations with histology and quantitative gene expression.

BACKGROUND AND AIMS: The advantage of combining molecular and morphological imaging, e.g. positron emission tomography and magnetic resonance imaging (PET/MRI), is reflected in the increased use of these modalities as surrogate end-points in clinical trials. This study aimed at evaluating plaque inflammation using 18F-fluorodeoxyglucose (18F-FDG)-PET/MRI, and gene expression in a minipig model of atherosclerosis. METHODS: Göttingen Minipigs were fed for 60 weeks with fat/fructose/cholesterol-rich diet (FFC), chow (Control) or FFC-diet changed to chow midway (diet normalization group; DNO). In all groups, 18F-FDG-PET/MRI of the abdominal aorta was assessed midway and at study-end. The aorta was analyzed using histology and gene expression. RESULTS: At study-end, FFC had significantly higher FDG-uptake compared to Control (target-to-background maximal uptake, TBRMax (95% confidence interval) CITBRMax: 0.092; 7.32) and DNO showed significantly decreased uptake compared to FFC (CITBRMax: -5.94;-0.07). No difference was observed between DNO and Control (CITBRMax: -2.71; 4.11). FFC displayed increased atherosclerosis and gene expression of inflammatory markers, including vascular cell adhesion molecule 1 (VCAM-1), cluster of differentiation 68 (CD68), matrix metalloproteinase 9 (MMP9), cathepsin K (CTSK) and secreted phosphoprotein 1 (SPP1) compared to Control and DNO (all, p < 0.05). FDG-uptake correlated with gene expression of inflammatory markers, including CD68, ρs = 0.58; MMP9, ρs = 0.46; SPP1, ρs = 0.44 and CTSK, ρs = 0.49; (p ≤ 0.01 for all). CONCLUSIONS: In a model of atherosclerosis, 18F-FDG-PET/MRI technology allows for detection of inflammation in atherosclerotic plaques, consistent with increased inflammatory gene expression. Our findings corroborate clinical data and are important in pre-clinical drug development targeting plaque inflammation.

Age- and Sex-Associated Effects on Acute-Phase Proteins in Göttingen Minipigs.

Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40-48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli.

Noninvasive assessment of pulse-wave velocity and flow-mediated vasodilation in anesthetized Göttingen minipigs.

Few methods for noninvasive assessment of arterial stiffness and endothelial dysfunction in porcine models are available. The aim of this study was to evaluate methods for assessment of arterial stiffness and endothelial dysfunction in anesthetized Göttingen minipigs. Pulse-wave velocity (PWV) was assessed in male Göttingen minipigs (n = 8; age approximately 60 wk) by using applanation tonometry of the carotid and femoral arteries. In addition, flow-mediated vasodilation (FMD) was assessed by using vascular ultrasonography of the brachial artery to evaluate endothelial dysfunction. To evaluate the reproducibility of the methods, minipigs were anesthetized by intravenous infusion of ketamine and midazolam and examined every other day for a total of 3 trials. Neither examination day nor systolic, diastolic, or mean arterial blood pressure statistically influenced PWV or FMD. The median interexamination coefficient of variation was 17% for PWV and 59% for FMD. Measured values of PWV corresponded largely to those in clinically healthy humans, but FMD values were lower than expected for lean, young animals. Although the ketamine-midazolam anesthesia we used has been associated with minor hemodynamic effects in vivo, in vitro studies suggest that both drugs are vasodilatory. Therefore anesthesia might have influenced the endothelial response, contributing to the modest FMD response and the concurrent high coefficients of variation that we noted. We conclude that PWV­but not FMD­showed acceptable interexamination variation for its potential application in porcine models.

Feasibility of simultaneous PET/MR in diet-induced atherosclerotic minipig: a pilot study for translational imaging.

Novel hybrid 18-fluoro-deoxy-D-glucose ((18)F-FDG) based positron emission tomography (PET) and magnetic resonance imaging (MRI) has shown promise for characterization of atherosclerotic plaques clinically. The purpose of this study was to evaluate the method in a pre-clinical model of diet-induced atherosclerosis, based on the Göttingen minipig. Using (18)F-FDG PET/MRI the goal was to develop and create a new imaging method in an in vivo animal model for translational studies of atherosclerosis. We used a strategy of multisequence MRI for optimal anatomical imaging of the abdominal aortas of the pigs (n=4): T1-weighted turbo spin-echo (T1-TSE), T2-weighted turbo spin-echo (T2-TSE) and proton density imaging with and without fat saturation. (18)F-FDG PET emission data were collected from a single bed position of the abdominal aorta in 3D mode for either 10 (n=4) or 10 and 20 minutes (n=2) to measure glycolysis as given by standardized uptake values (SUV). Ex vivo en face evaluation of aortas from an atherosclerotic animal illustrated plaque distribution macroscopically, compared to a lean control animal. Although T2-TSE weighted imaging was most consistent, no one MRI sequence was preferable and superior to another for visualization and identification of the abdominal aorta. We found poor correlation between SUVs obtained from 10 and 20 minutes of reconstructed PET emission data. This can most likely be ascribed to intestinal movement. In conclusion multisequence MRI is recommended for optimal imaging of the abdominal aorta using MRI. Furthermore we found that 10 minutes of PET emission data seems adequate. This is the first study to demonstrate that the method of (18)F-FDG PET/MRI is feasible in minipig models of atherosclerosis, and therefore relevant in larger prospective studies. Perspectives of the method include correlation to e.g. aortic immunohistochemistry findings and a range of genomic and proteomic analyses.

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.

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.

Orosomucoid expression profiles in liver, adipose tissues and serum of lean and obese domestic pigs, Göttingen minipigs and Ossabaw minipigs.

The acute phase protein orosomucoid (ORM) has anti-inflammatory and immunomodulatory effects, and may play an important role in the maintenance of metabolic homeostasis in obesity-induced low-grade inflammation. Even though the pig is a widely used model for obesity related metabolic symptoms, the expression of ORM has not yet been characterized in such pig models. The objective of this study was to investigate the expression of ORM1 mRNA in liver, visceral adipose tissue, subcutaneous adipose tissue (SAT) from the abdomen or retroperitoneal abdominal adipose tissue (RPAT) and SAT from the neck, as well as the serum concentration of ORM protein in three porcine obesity models; the domestic pig, Göttingen minipigs and Ossabaw minipigs. No changes in ORM1 mRNA expression were observed in obese pigs compared to lean pigs in the four types of tissues. However, obese Ossabaw minipigs, but none of the other breeds, showed significantly elevated ORM serum concentrations compared to their lean counterparts. Studies in humans have shown that the expression of ORM was unchanged in adipose tissue depots in obese humans with an increased serum concentration of ORM. Thus in this respect, obese Ossabaw minipigs behave more similarly to obese humans than the other two pig breeds investigated.