Nanopore sequencing reveals methylation changes associated with obesity in circulating cell-free DNA from Göttingen Minipigs.

Profiling of circulating cell-free DNA (cfDNA) by tissue-specific base modifications, such as 5-methylcytosines (5mC), may enable the monitoring of ongoing pathophysiological processes. Nanopore sequencing allows genome-wide 5mC detection in cfDNA without bisulphite conversion. The aims of this study were: i) to find differentially methylated regions (DMRs) of cfDNA associated with obesity in Göttingen minipigs using Nanopore sequencing, ii) to validate a subset of the DMRs using methylation-specific PCR (MSP-PCR), and iii) to compare the cfDNA DMRs with those from whole blood genomic DNA (gDNA). Serum cfDNA and gDNA were obtained from 10 lean and 7 obese Göttingen Minipigs both with experimentally induced myocardial infarction and sequenced using Oxford Nanopore MinION. A total of 1,236 cfDNA DMRs (FDR<0.01) were associated with obesity. In silico analysis showed enrichment of the adipocytokine signalling, glucagon signalling, and cellular glucose homoeostasis pathways. A strong cfDNA DMR was discovered in PPARGC1B, a gene linked to obesity and type 2 diabetes. The DMR was validated using MSP-PCR and correlated significantly with body weight (P < 0.05). No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA. In conclusion, nanopore sequencing detected differential methylation in minute quantities (0.1-1 ng/µl) of cfDNA. Future work should focus on translation into human and comparing 5mC from somatic tissues to pinpoint the exact location of pathology.

Oxford nanopore sequencing can reveal changes in methylation patterns associated with obesity in minute quantities of cell-free DNA from serum.Bisulphite conversion and methylation-specific PCR can be used to validate differentially methylated regions in cell-free DNA.A differentially methylated region in an intronic region of the PPARGC1B gene was found associated with obesity.Differentially methylated regions in cell-free DNA could be useful as early risk markers of certain diseases and pathologies.

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.

Optimization of pig models for translation of subcutaneous pharmacokinetics of therapeutic proteins: Liraglutide, insulin aspart and insulin detemir.

Prediction of human pharmacokinetics (PK) from data obtained in animal studies is essential in drug development. Here, we present a thorough examination of how to achieve good pharmacokinetic data from the pig model for translational purposes by using single-species allometric scaling for selected therapeutic proteins: liraglutide, insulin aspart and insulin detemir. The predictions were based on non-compartmental analysis of intravenous and subcutaneous PK data obtained from two injection regions (neck, thigh) in two pig breeds, domestic pig and Göttingen Minipig, that were compared with PK parameters reported in humans. The effects of pig breed, injection site and injection depth (insulin aspart only) on the PK of these proteins were also assessed. Results show that the prediction error for human PK was within two-fold for most PK parameters in both pig breeds. Furthermore, pig breed significantly influenced the plasma half-life and mean absorption time (MAT), both being longer in Göttingen Minipigs compared to domestic pigs (P <0.01). In both breeds, thigh vs neck dosing was associated with a higher dose-normalized maximum plasma concentration and area under the curve as well as shorter MAT and plasma half-life (P <0.01). Finally, more superficial injections resulted in faster absorption, higher Cmax/dose and bioavailability of insulin aspart (P <0.05, 3.0 vs 5.0 mm injection depth). In conclusion, pig breed and injection region affected the PK of liraglutide, insulin aspart and insulin detemir and reliable predictions of human PK were demonstrated when applying single-species allometric scaling with the pig as a pre-clinical animal model.

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 Angiogenesis PET/MR in a Porcine Model of Chronic Myocardial Infarction.

Angiogenesis is crucial in tissue repair and prevents scar tissue formation following an ischemic event such as myocardial infarction. The ischemia induces formation of new capillaries, which have high expression of integrin αvß3. [68Ga]Ga-NODAGA-E[(cRGDyK)]2 ([68Ga]Ga-RGD) is a promising PET-radiotracer reflecting angiogenesis by binding to integrin αvß3. A Göttingen mini-pig underwent transient catheter-induced left anterior descending artery (LAD) occlusion for 120 min, and after 8 weeks was imaged on a Siemens mMR 3T PET/MR system. A large antero-septal infarction was evident by late gadolinium enhancement (LGE) on the short axis and 2-4 chamber views. The infarcted area corresponded to the area with high [68Ga]Ga-RGD uptake on the fused PET/MR images, with no uptake in the healthy myocardium. To support the hypothesis that [68Ga]Ga-RGD uptake reflects angiogenesis, biopsies were sampled from the infarct border and healthy myocardium. Expression of αvß3 was evaluated using immunohistochemistry. The staining showed higher αvß3 expression in the capillaries of the infarct border compared to those in the healthy myocardium. These initial data confirm in vivo detection of angiogenesis using [68Ga]Ga-RGD PET in a translational model, which overall support the method applicability when evaluating novel cardio-protective therapies.

Atorvastatin impairs liver mitochondrial function in obese Göttingen Minipigs but heart and skeletal muscle are not affected.

Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.

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.

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.

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.

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.