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.

Experimental non-alcoholic steatohepatitis in Göttingen Minipigs: consequences of high fat-fructose-cholesterol diet and diabetes.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in humans, and ranges from steatosis to non-alcoholic steatohepatitis (NASH), the latter with risk of progression to cirrhosis. The Göttingen Minipig has been used in studies of obesity and diabetes, but liver changes have not been described. The aim of this study was to characterize hepatic changes in Göttingen Minipigs with or without diabetes, fed a diet high in fat, fructose, and cholesterol to see if liver alterations resemble features of human NAFLD/NASH. METHODS: Fifty-four male castrated minipigs (age 6 to 7 months) were distributed into four groups and diet-fed for 13 months. Groups were: lean controls fed standard diet (SD, n = 8), a group fed high fat/fructose/cholesterol diet (FFC, n = 16), a group fed high fat/fructose/cholesterol diet but changed to standard diet after 7 months (diet normalization, FFC/SD, n = 16), and a streptozotocin-induced diabetic group fed high fat/fructose/cholesterol diet (FFCDIA, n = 14). At termination, blood samples for analyses of circulating biomarkers and liver tissue for histopathological assessment and analyses of lipids and glycogen content were collected. RESULTS: In comparison with SD and FFC/SD, FFC and FFCDIA pigs developed hepatomegaly with increased content of cholesterol, whereas no difference in triglyceride content was found. FFC and FFCDIA groups had increased values of circulating total cholesterol and triglycerides and the hepatic circulating markers alkaline phosphatase and glutamate dehydrogenase. In the histopathological evaluation, fibrosis (mainly located periportally) and inflammation along with cytoplasmic alterations (characterized by hepatocytes with pale, granulated cytoplasm) were found in FFC and FFCDIA groups compared to SD and FFC/SD. Interestingly, FFC/SD also had fibrosis, a feature not seen in SD. Only two FFC and three FFCDIA pigs had > 5% steatosis, and no hepatocellular ballooning or Mallory-Denk bodies were found in any of the pigs. CONCLUSIONS: Fibrosis, inflammation and cytoplasmic alterations were characteristic features in the livers of FCC and FFCDIA pigs. Overall, diabetes did not exacerbate the hepatic changes compared to FFC. The limited presence of the key human-relevant pathological hepatic findings of steatosis and hepatocellular ballooning and the variation in the model, limits its use in preclinical research without further optimisation.

Hyperglycemia-induced transcriptional regulation of ROCK1 and TGM2 expression is involved in small artery remodeling in obese diabetic Göttingen Minipigs.

Obesity and diabetes in humans are associated with hypertrophic remodeling and increased media:lumen ratio of small resistance arteries, which is an independent predictor of cardiovascular events. In order to minimize increases in media:lumen ratio, hypertrophic remodeling should be accompanied by outward remodeling. We aimed to investigate the mechanisms of structural remodeling in small pial arteries (PAs) and terminal mesenteric arteries (TMAs) from obese Göttingen Minipigs with or without diabetes. Göttingen Minipigs received either control diet (lean control (LC)), high fat/high fructose/high cholesterol diet (FFC), or FFC diet with streptozotocin (STZ)-induced diabetes (FFC/STZ) for 13 months. At the end of the study (20 months), we assessed body weight, fasting plasma biochemistry, passive vessel dimensions, mRNA expression (matrix metallopeptidases 2/9 (MMP2, MMP9), tissue inhibitor of metallopeptidase 1 (TIMP1), transglutaminase 2 (TGM2), Rho-kinase 1 (ROCK1), TGFß-receptor 2 (TGFBR2), and IGF1-receptor (IGFR1) genes), and immunofluorescence in PAs and TMAs. We performed multiple linear correlation analyses using plasma values, structural data, and gene expression data. We detected outward hypertrophic remodeling in TMAs and hypertrophic remodeling in PAs from FFC/STZ animals. ROCK1 and TGM2 genes were up-regulated in PAs and TMAs from the FFC/STZ group. Passive lumen diameter (PLD) of TMAs was correlated with plasma values of glucose (GLU), fructosamine (FRA), total cholesterol (TC), and triglycerides (TGs). ROCK1 and TGM2 expressions in TMAs were correlated with PLD, plasma GLU, fructosamine, and TC. ROCK1 and TGM2 proteins were immunolocalized in the media of PAs and TMAs, and their fluorescence levels were increased in the FFC/STZ group. Hyperglycemia/hyperlipidemia is involved in regulation of ROCK1 and TGM2 expression leading to outward remodeling of small resistance arteries in obese diabetic Göttingen Minipigs.

Nonclinical evaluation of immunological safety in Göttingen Minipigs: The CONFIRM initiative.

There is a growing need to consider non-rodent species for the immunological safety evaluation of drug candidates. The EU Framework-6 RETHINK Project demonstrated that the Göttingen Minipig is a relevant animal model for regulatory toxicology studies. Extensive knowledge on the immune system of domestic pigs is available and fewer differences from humans have been identified as compared to other species, such as mice or non-human primates. Minipig data are too scarce to allow for claiming full immunological comparability with domestic pigs. Another gap limiting minipig use for immunological safety evaluation is the lack of a qualified and validated database. However, available data lend support to the use of minipigs. The need for a COllaborative Network For Immunological safety Research in Minipigs (the CONFIRM Initiative) was obvious. It is intended to trigger immunological safety research in Göttingen Minipigs, to assist and synergize fundamental, translational and regulatory investigative efforts relevant to the immunological safety evaluation of pharmaceuticals and biologics, and to spread current knowledge and new findings to the scientific and regulatory toxicology community.

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Göttingen minipig model of diet-induced atherosclerosis: influence of mild streptozotocin-induced diabetes on lesion severity and markers of inflammation evaluated in obese, obese and diabetic, and lean control animals.

BACKGROUND: From a pharmacological perspective, readily-available, well-characterized animal models of cardiovascular disease, including relevant in vivo markers of atherosclerosis are important for evaluation of novel drug candidates. Furthermore, considering the impact of diabetes mellitus on atherosclerosis in human patients, inclusion of this disease aspect in the characterization of a such model, is highly relevant. The objective of this study was to evaluate the effect of mild streptozotocin-induced diabetes on ex- and in vivo end-points in a diet-induced atherosclerotic minipig model. METHODS: Castrated male Göttingen minipigs were fed standard chow (CD), atherogenic diet alone (HFD) or with superimposed mild streptozotocin-induced diabetes (HFD-D). Circulating markers of inflammation (C-reactive protein (CRP), oxidized low-density lipoprotein (oxLDL), plasminogen activator inhibitor-1, lipid and glucose metabolism were evaluated together with coronary and aortic atherosclerosis after 22 or 43 diet-weeks. Group differences were evaluated by analysis of variance for parametric data and Kruskal-Wallis test for non-parametric data. For qualitative assessments, Fisher's exact test was applied. For all analyses, p < 0.05 was considered statistically significant. RESULTS: Overall, HFD and HFD-D displayed increased CRP, oxLDL and lipid parameters compared to CD at both time points. HFD-D displayed impaired glucose metabolism as compared to HFD and CD. Advanced atherosclerotic lesions were observed in both coronary arteries and aorta of HFD and HFD-D, with more advanced plaque findings in the aorta but without differences in lesion severity or distribution between HFD and HFD-D. Statistically, triglyceride was positively (p = 0.0039), and high-density lipoprotein negatively (p = 0.0461) associated with aortic plaque area. CONCLUSIONS: In this model, advanced coronary and aortic atherosclerosis was observed, with increased levels of inflammatory markers, clinically relevant to atherosclerosis. No effect of mild streptozotocin-induced diabetes was observed on plaque area, lesion severity or inflammatory markers.

High fat diet is associated with gut microbiota dysbiosis and decreased gut microbial derived metabolites related to metabolic health in young Göttingen Minipigs.

The objectives were 1) to characterize a Göttingen Minipig model of metabolic syndrome regarding its colon microbiota and circulating microbial products, and 2) to assess whether ovariectomized female and castrated male minipigs show similar phenotypes. Twenty-four nine-week-old Göttingen Minipigs were allocated to four groups based on sex and diet: ovariectomized females and castrated males fed either chow or high-fat diet (HFD) for 12 weeks. At study end, body composition and plasma biomarkers were measured, and a mixed meal tolerance test (MMT) and an intravenous glucose tolerance test (IVGTT) were performed. The HFD groups had significantly higher weight gain, fat percentage, fasting plasma insulin and glucagon compared to the chow groups. Homeostatic model assessment of insulin resistance index (HOMA-IR) was increased and glucose effectiveness derived from the IVGTT and Matsuda´s insulin sensitivity index from the MMT were decreased in the HFD groups. The HFD groups displayed dyslipidemia, with significantly increased total-, LDL- and HDL-cholesterol, and decreased HDL/non-HDL cholesterol ratio. The colon microbiota of HFD minipigs clearly differed from the lean controls (GuniFrac distance matrix). The main bacteria families driving this separation were Clostridiaceae, Fibrobacteraceae, Flavobacteriaceae and Porphyromonadaceae. Moreover, the species richness was significantly decreased by HFD. In addition, HFD decreased the circulating level of short chain fatty acids and beneficial microbial metabolites hippuric acid, xanthine and trigonelline, while increasing the level of branched chain amino acids. Six and nine metabolically relevant genes were differentially expressed between chow-fed and HFD-fed animals in liver and omental adipose tissue, respectively. The HFD-fed pigs presented with metabolic syndrome, gut microbial dysbiosis and a marked decrease in healthy gut microbial products and thus displayed marked parallels to human obesity and insulin resistance. HFD-fed Göttingen Minipig therefore represents a relevant animal model for studying host-microbiota interactions. No significant differences between the castrated and ovariectomized minipigs were observed.

Functional and morphological renal changes in a Göttingen Minipig model of obesity-related and diabetic nephropathy.

Obesity-related glomerulopathy and diabetic nephropathy (DN) are serious complications to metabolic syndrome and diabetes. The purpose was to study effects of a fat, fructose and cholesterol-rich (FFC) diet with and without salt in order to induce hypertension on kidney function and morphology in Göttingen Minipigs with and without diabetes. Male Göttingen Minipigs were divided into 4 groups: SD (standard diet, n = 8), FFC (FFC diet, n = 16), FFC-DIA (FFC diet + diabetes, n = 14), FFC-DIA + S (FFC diet with extra salt + diabetes, n = 14). Blood and urine biomarkers, glomerular filtration rate (GFR), blood pressure (BP) and resistive index (RI) were evaluated after 6-7 months (T1) and 12-13 months (T2). Histology, electron microscopy and gene expression (excluding FFC-DIA + S) were evaluated at T2. All groups fed FFC-diet displayed obesity, increased GFR and RI, glomerulomegaly, mesangial expansion (ME) and glomerular basement membrane (GBM) thickening. Diabetes on top of FFC diet led to increased plasma glucose and urea and proteinuria and tended to exacerbate the glomerulomegaly, ME and GBM thickening. Four genes (CDKN1A, NPHS2, ACE, SLC2A1) were significantly deregulated in FFC and/or FFC-DIA compared to SD. No effects on BP were observed. Göttingen Minipigs fed FFC diet displayed some of the renal early changes seen in human obesity. Presence of diabetes on top of FFC diet exacerbated the findings and lead to changes resembling the early phases of human DN.

Implantation of telemetric blood pressure transmitters in Göttingen Minipigs: Validation of 24-h systemic blood pressure and heart rate monitoring and influence of anaesthesia.

INTRODUCTION: Porcine animal models are used in biomedical research due to anatomical and physiological similarities with human patients. The study aimed to validate telemetric systemic blood pressure (BP) and heart rate (HR) monitoring in Göttingen Minipigs, and in addition to study the effects of three different anaesthesia protocols on telemetric BP and HR measurements. METHODS: Eight female Göttingen Minipigs had telemetry transmitters implanted in the right carotid artery. Over ten weeks, systemic 24-h BP and HR monitoring were repeated four times, each ending with an angiotensin II stimulation test. In addition, systemic BP and HR evaluated by telemetry, intra-arterial catheter (IAC) and oscillometric tail-cuff were compared before and after the 10-weeks period. Furthermore, changes in telemetric systemic BP and HR were monitored during anaesthesia in a cross-over design using three different protocols of general anaesthesia: Midazolam/ketamine (MK), propofol, and a combination of tiletamine, zolazepam, xylazine, ketamine and butorphanol (Zoletil-mix). RESULTS: One minipig was excluded and some data were missing due to central-venous catheter issues. The coefficient of variation was below 10% for the 24-h BP and HR measurements, but higher during angiotensin II stimulation. There was a disagreement between the tail-cuff measurement and telemetry/IAC, however the differences were independent of the BP and HR level. All anaesthesia protocols numerically influenced BP and HR, but only propofol statistically significantly decreased the BP. CONCLUSION: The study showed acceptable reproducibility of telemetric measurement of BP and HR over ten weeks in freely moving Göttingen Minipigs. There was a disagreement between direct and indirect BP measurement, and BP and HR were influenced by all anaesthesia protocols.

Dietary normalization from a fat, fructose and cholesterol-rich diet to chow limits the amount of myocardial collagen in a Göttingen Minipig model of obesity.

BACKGROUND: Dietary interventions have been shown to attenuate some of the myocardial pathological alterations associated with obesity. This study evaluated the effect of dietary normalization from a fat/fructose/cholesterol-rich diet to chow on left ventricular (LV) myocardial fibrosis, fat infiltration and hypertrophy but also the specific influence of obesity, plasma lipids and glucose metabolism markers on heart morphology in a Göttingen Minipig model of obesity. METHODS: Forty castrated male Göttingen Minipigs were assigned to three groups fed either standard minipig chow (SD, n = 8) for 13 months, fat/fructose/cholesterol-rich diet (FFC, n = 16) for 13 months or fat/fructose/cholesterol-rich diet for 7 months and then changed to standard minipig chow for the remaining 6 months (FFC/SD, n = 16). Body weight, body fat percentage, plasma lipids and glucose metabolism markers were evaluated in all three groups after 6-7 months (prior to diet adjustment for FFC/SD) and again before termination. Further, biochemical quantification of myocardial collagen and triglyceride content, semi-quantitative histological evaluation of fibrosis and fat infiltration and quantitative histological analysis of collagen and cardiomyocyte diameter were performed and heart weight was obtained after termination. Group differences were evaluated using Kruskal-Wallis test and Fisher's exact test for categorical variables. Pearson correlation analysis was performed to test for correlations between myocardial changes and selected explanatory variables. For non-parametric response variables, a Spearman correlation analysis was applied. RESULTS: Myocardial collagen content quantified biochemically was significantly lower in FFC/SD compared to FFC (P = 0.02). Furthermore, dietary normalization from a fat/fructose/cholesterol-rich diet to chow caused stagnation of body weight and body fat percentage, normalized intravenous glucose tolerance index (KG) and plasma lipid levels. CONCLUSION: Dietary normalization led to lower LV collagen content in obese Göttingen Minipigs. Despite gross obesity and significant deteriorations in glucose and lipid metabolism, only mild myocardial changes were found in this model of obesity and therefore further model optimization is warranted in order to induce more severe myocardial changes before dietary or pharmacological interventions.

Neurohormonal and circulatory effects of short-term treatment with enalapril and quinapril in dogs with asymptomatic mitral regurgitation.

The aim of the study was to compare the effect of 2 angiotensin-converting enzyme (ACE) inhibitors on neurohormonal and circulatory variables in Cavalier King Charles Spaniels (CKCSs) with asymptomatic mitral regurgitation (MR). Ten CKCSs with mild to severe untreated MR were treated with 2 ACE inhibitors, quinapril and enalapril (each at 0.5 mg/kg PO q24h for 7 days), in a double-blind, crossover study with a washout period of 7 days between treatments. Blood samples were drawn and echocardiography was performed on days 0, 7, 14, and 21. Both treatments reduced ACE activity (P < .001) and increased renin activity (P < .001) and atrial natriuretic peptide concentration (P < .005). The ACE inhibitors had no effect on the concentrations of nitrate and nitrite (NOx) or asymmetric dimethylarginine (ADMA). On day 0, a lower NOx concentration (P = .02) was found in samples taken in the clinic as compared to samples taken in the homes of the dogs. Quinapril caused a significant reduction in more variables that reflect the severity of MR (eg, jet size and left ventricular end diastolic diameter) than did enalapril. However, in terms of specific variables, no significant difference was identified between the effects of the 2 treatments on MR. These results suggest that ACE inhibitors do not affect NOx and ADMA concentrations in asymptomatic dogs, but exercise, stress, or some combination may influence NOx concentrations in these dogs.

Efficacy of enalapril for prevention of congestive heart failure in dogs with myxomatous valve disease and asymptomatic mitral regurgitation.

We evaluated the long-term effect of early angiotensin-converting enzyme (ACE) inhibition (enalapril maleate) as monotherapy to postpone or prevent congestive heart failure (CHF) in asymptomatic dogs with mitral regurgitation (MR) attributable to myxomatous valvular disease (MVD) in a prospective, randomized, double-blinded, placebo-controlled multicenter trial involving 14 centers in Scandinavia. Two hundred twenty-nine Cavalier King Charles (CKC) Spaniels with MR attributable to MVD but no signs of CHF were randomly allocated to treatment with enalapril 0.25-0.5 mg daily (n = 116) or to placebo groups (n = 113). Each dog was evaluated by physical examination, electrocardiography, and thoracic radiography at entry and every 12 months (+/-30 days). The number of dogs developing heart failure was similar in the treatment and placebo groups (n = 50 [43%] and n = 48 [42%], respectively; P = .99). The estimated means, adjusted for censored observations, for the period from initiation of therapy to heart failure were 1,150 +/- 50 days for dogs in the treatment group and 1,130 +/- 50 days for dogs in the placebo group (P = .85). When absence or presence of cardiomegaly at the entrance of the trial was considered, there were still no differences between the treatment and placebo groups (P = .98 and .51, respectively). Multivariate analysis showed that enalapril had no significant effect on the time from initiation of therapy to heart failure (P = .86). Long-term treatment with enalapril in asymptomatic dogs with MVD and MR did not delay the onset of heart failure regardless of whether or not cardiomegaly was present at initiation of the study.

Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease.

OBJECTIVES: To evaluate the predictive value of plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) and nitric oxide end-products (NOx) as markers for progression of mitral regurgitation caused by myxomatous mitral valve disease. ANIMALS: Seventy-eight privately owned Cavalier King Charles spaniels with naturally occurring myxomatous mitral valve disease. METHODS: Prospective longitudinal study comprising 312 measurements over a 4.5 year period. Clinical values were recorded, NT-proANP concentrations were measured by radioimmunoassay, and NOx were analyzed colorimetrically. To predict congestive heart failure (CHF), Cox proportional hazards models with time-varying covariates were constructed. RESULTS: The hazard ratio for NT-proANP (per 1000 pmol/l increase) to predict future CHF was 6.7 (95% confidence interval, 3.6-12.5; p < 0.001). The median time to CHF for dogs with NT-proANP levels >1000 pmol/l was 11 months (95% confidence interval, 5.6-12.6 months), compared to 54 months (46 - infinity) for dogs with concentrations ≤ 1000 pmol/l (p < 0.001). Due to intra- and inter-individual variability, most corresponding analyses for NOx were insignificant but dogs reaching CHF had a lower mean NOx concentration than dogs not reaching CHF (23 vs. 28 µmol/l, p = 0.016). Risk of CHF increased with increase in heart rate (>130 beats per minute) and grade of murmur (≥ 3/6). CONCLUSIONS: The risk of CHF due to mitral regurgitation is increased in dogs with blood NT-proANP concentrations above 1000 pmol/l. Measurement of NT-proANP can be a valuable tool to identify dogs that may develop CHF within months.