To understand the mechanisms underlying the remarkable resistance of Göttingen minipigs to metabolic dysfunction-associated steatotic liver disease (MASLD), a multilevel transcriptomic analysis comparing their liver with those of humans, mice, and MASLD susceptible pig breeds was performed. This analysis revealed 692 genes uniquely differentially expressed in livers of Göttingen minipigs, linked to multiple metabolic and signaling pathways (e.g., AMPK signaling). Among these, 11 transcription factors (TFs), 3 hepatokines (DPP4, ITIH1, and ITIH3), and 6 additional secreted proteins (including PCSK9 and APOM) exhibited particularly strong differential expression and emerged as candidate mediators of MASLD resistance. This finding was further supported by weighted gene coexpression network analysis, which identified a core regulatory network driven by the TFs HMBOX1 and PATZ1, presumably regulating 41% of its differentially expressed genes. Collectively, these results suggest that a distinct transcriptional program involving HMBOX1/PATZ1 and their downstream targets, including secreted factors, contributes to the MASLD-resistant phenotype of Göttingen minipigs and may provide promising targets for the prevention and treatment of fatty liver disease.

The vascular system ensures sufficient blood supply and tissue homeostasis and consists of different cell types. Endothelial cells represent the structural backbone of blood vessels and are accompanied by mural cells, specifically pericytes in the microcirculation as well as vascular smooth muscle cells (vSMC) along the larger vessels (arteries, arterioles and veins). Distinguishing these different cell types in immunohistochemical stainings presents a challenge due to their close proximity and the unreliable marker distribution of mural cells. Furthermore, manual quantification of capillaries and pericytes is highly examiner-dependent, hindering inter-examiner and inter-laboratory comparisons. To address these issues, we developed an automated algorithm-based analysis software designed to standardize quantification of vascular structures in immunohistochemical images, named CAPPER (Capillary / Pericyte Quantification Tool). Through the implementation of adaptive thresholding, morphological operations, and domain-specific knowledge, CAPPER excels in the quantification of capillary densities and pericyte coverage in different organs (brain, heart, muscle, and kidney), species (mouse and pig) as well as a plethora of disease states. We furthermore propose a robust pericyte marker array to more accurately identify this elusive cell type.

The translation of novel discoveries in biomedical research into clinical applications is a lengthy and resource-intensive process, with variable success. Predictive model organisms for non-clinical research are a key asset in this process. Rodents remain the most widely used models, but they have limitations in recapitulating human pathophysiology and disease phenotypes. Over the past two decades, pigs have played an increasingly important role in bridging this translational gap. Given their close anatomical and physiological similarity to humans, most vividly illustrated by successful pig-to-human organ xenotransplantation, pigs provide unparalleled advantages for translational research. Advances in genetic engineering now allow the precise recreation of human disease mechanisms in pigs, generating tailored large-animal models with exceptional clinical relevance. This review highlights the potential of genetically modified pig models for human diseases, with a focus on metabolic disorders and rare monogenic diseases such as Duchenne muscular dystrophy, cystic fibrosis, and Laron syndrome.

Both diabetes mellitus (DM) and chronic kidney disease (CKD) predispose to cardiac remodeling and coronary microvascular dysfunction, which is proposed to be mediated through increased oxidative stress. To link oxidative stress and cardiac remodeling in DM and CKD, CKD was induced in genetically modified DM swine ( INS C94Y transgenic) at 10–12 weeks of age via renal microembolization, while non-embolized DM and wild-type (WT) swine served as controls. Compared to WT, 1) DM animals displayed modest LV dilation and a slight decline in ejection fraction, with increased end-systolic pressures and coronary blood flow. Addition of CKD did not further aggravate these alterations, but increased the pressure and diastolic wall stress compared to WT. 2) Proteomic analysis revealed enrichment in metabolic pathways involving fatty acids and glutamate, thus highlighting substantial metabolic reprogramming in both DM and DM_CKD groups. Re-analysis of proteomic data from human HFpEF patients showed differential regulation of similar pathways as well as anti-oxidant enzymes. 3) Isolated mitochondrial respiration was reduced in DM and DM_CKD hearts across multiple substrates (fatty acids, pyruvate-malate, glutamate-malate, and succinate), implicating broad mitochondrial dysfunction. 4) DM_CKD animals showed heightened oxidative stress in both coronary vasculature and myocardium as compared to both WT and DM [13 ± 3 (WT), 14 ± 3 (DM) and 39 ± 10% positive nuclei (DM_CKD)]. 5) LV-interstitial fibrosis was increased in DM_CKD (3.72 ± 0.50%) vs WT (1.70 ± 0.29%), with DM having an intermediate phenotype (2.82 ± 0.37%). Thus, even mild CKD in the presence of DM is accompanied by oxidative stress and ECM deposition. Our findings highlight the critical role of CKD in accelerating cardiac pathology and underscore the importance of targeting the cardiorenal axis in future therapeutic strategies.

The pig is a valuable animal model in diabetes research; however, standardized protocols are essential for evaluating in vivo metabolism. Here, we present a protocol for in vivo assessment of glucose control and insulin secretion and sensitivity in the pig. We describe steps for catheter implantation, testing of intravenous glucose tolerance, performance of hyperinsulinemic-euglycemic clamps (HECs) and hyperglycemic clamps (HGCs), and blood processing. We then detail procedures for analysis of plasma glucose, insulin, glucagon, and C-peptide concentrations as well as data analysis. For complete details on the use and execution of this protocol, please refer to Renner et al.1 and Renner et al.2.

BACKGROUND AND AIMS:Fasting hypoglycemia has clinical implications for children with growth hormone (GH)-insensitivity syndrome. This study investigates the pathophysiology of juvenile hypoglycemia in a large animal model for GH receptor (GHR) deficiency (the GHR-KO pig) and elucidates mechanisms underlying the transition to normoglycemia in adulthood. METHODS:Insulin sensitivity was assessed in juvenile and adult GHR-KO pigs and wild-type (WT) controls via hyperinsulinemic-euglycemic clamp (HEC) tests. Glucose turnover was measured using D-[6,6-2H2] glucose and 2H2O. Clinical chemical and targeted metabolomics parameters in blood serum were correlated with qPCR and western blot analyses of liver and adipose tissue. RESULTS:GHR-KO pigs showed increased insulin sensitivity (p = 0.0019), especially at young age (M-value +34% vs. WT), insignificantly reduced insulin levels, and reduced endogenous glucose production (p = 0.0007), leading to fasting hypoglycemia with depleted liver glycogen, elevated β-hydroxybutyrate, but no increase in NEFA levels. Low hormone-sensitive lipase phosphorylation in adipose tissue suggested impaired lipolysis in young GHR-KO pigs. Metabolomics indicated enhanced fatty acid beta-oxidation and use of glucogenic amino acids, likely serving as compensatory pathways to maintain energy homeostasis. In adulthood, insulin sensitivity remained elevated but less pronounced (M-value +20%), while insulin levels were significantly reduced, enabling normoglycemia and improved NEFA availability. Increased fat mass, but not sex hormones, appeared key to this metabolic transition, as early castration had no effect. CONCLUSIONS:Juvenile hypoglycemia in GH insensitivity results from excessive insulin sensitivity, reduced glucose production, and impaired lipolysis. Normoglycemia in adulthood emerges through increased adiposity and moderated insulin sensitivity, independently of sex hormones. These findings elucidate the age-dependent metabolic adaptations in GH insensitivity.

Primary human pancreatic ductal organoids (HPDO) have emerged as a model to study pancreas biology and model disease like pancreatitis and pancreatic cancer. Yet, donor material availability, genetic variability and a lack of extensive benchmarking to healthy and disease pancreas limits the range of applications. To address this gap, we established porcine pancreatic ductal organoids (PPDO) as a system from a reliable, genetically defined and easily obtainable source to model pancreatic ductal/progenitor biology. We benchmarked PPDO to HPDO and primary porcine pancreas using single-cell RNA sequencing (scRNA-Seq). We observed no overt phenotypic differences in PPDO derived from distinct developmental stages using extensive proteomics profiling, with a WNT/basal cell signaling enriched population characterizing PPDO. PPDO exhibited differentiation potential towards mature ductal cells and limited potential towards endocrine lineages. We used PPDO as a chemical screening platform to assess the safety of FDA-approved drugs and showed conserved toxicity of statins and α-adrenergic receptor inhibitors between PPDO and HPDO cultures. Overall, our results highlight the PPDO as a model for mammalian duct/progenitor applications.

Growing evidence shows that the lung is an organ prone to injury by diabetes mellitus. However, the molecular mechanisms of these pulmonary complications have not yet been characterized comprehensively. To systematically study the effects of insulin deficiency and hyperglycaemia on the lung, we combined proteomics and lipidomics with quantitative histomorphological analyses to compare lung tissue samples from a clinically relevant pig model for mutant INS gene-induced diabetes of youth (MIDY) with samples from wild-type littermate controls. Among others, the level of pulmonary surfactant-associated protein A (SFTPA1), a biomarker of lung injury, was moderately elevated. Furthermore, key proteins related to humoral immune response and extracellular matrix organization were significantly altered in abundance. Importantly, a lipoxygenase pathway was dysregulated as indicated by 2.5-fold reduction of polyunsaturated fatty acid lipoxygenase ALOX15 levels, associated with corresponding changes in the levels of lipids influenced by this enzyme. Our multi-omics study points to an involvement of reduced ALOX15 levels and an associated lack of eicosanoid switching as mechanisms contributing to a proinflammatory milieu in the lungs of subjects with diabetes mellitus.

Allogeneic intraportal islet transplantation (ITx) has become an established treatment for patients with poorly controlled type 1 diabetes. However, the loss of viable beta-cell mass after transplantation remains a major challenge. Therefore, noninvasive imaging methods for long-term monitoring of the transplant fate are required. In this study, [68Ga]Ga-DOTA-exendin-4 positron emission tomography/computed tomography (PET/CT) was used for repeated monitoring of allogeneic neonatal porcine islets (NPI) after intraportal transplantation into immunosuppressed genetically diabetic pigs. NPI transplantation (3320-15,000 islet equivalents per kg body weight) led to a reduced need for exogenous insulin therapy and finally normalization of blood glucose levels in 3 out of 4 animals after 5 to 10 weeks. Longitudinal PET/CT measurements revealed a significant increase in standard uptake values in graft-bearing livers. Histologic analysis confirmed the presence of well-engrafted, mature islet clusters in the transplanted livers. Our study presents a novel large animal model for allogeneic intraportal ITx. A relatively small dose of NPIs was sufficient to normalize blood glucose levels in a clinically relevant diabetic pig model. [68Ga]Ga-DOTA-exendin-4 PET/CT proved to be efficacious for longitudinal monitoring of islet transplants. Thus, it could play a crucial role in optimizing ITx as a curative therapy for type 1 diabetes.

INSC94Y transgenic pigs represent a model for mutant insulin gene-induced diabetes of youth, with impaired insulin secretion and beta cell loss, leading to elevated fasting blood glucose levels. A key complication of diabetes mellitus is diabetic retinopathy (DR), characterized by hyperglycemia-induced abnormalities in the retina. Adjacent to the retina lies the vitreous, a gelatinous matrix vital for ocular function. It harbors proteins and signaling molecules, offering insights into vitreous biology and ocular health. Moreover, as a reservoir for secreted molecules, the vitreous illuminates molecular processes within intraocular structures, especially under pathological conditions. To uncover the proteomic profile of porcine vitreous and explore its relevance to DR, we employed discovery proteomics to compare vitreous samples from INSC94Y transgenic pigs and wild-type controls. Our analysis identified 1404 proteins, with 266 showing differential abundance in INSC94Y vitreous. Notably, the abundances of ITGB1, COX2, and GRIFIN were significantly elevated in INSC94Y vitreous. Gene Set Enrichment Analysis unveiled heightened MYC and mTORC1 signaling in INSC94Y vitreous, shedding light on its biological significance in diabetes-associated ocular pathophysiology. These findings deepen our understanding of vitreous involvement in DR and provide valuable insights into potential therapeutic targets. Raw data are accessible via ProteomeXchange (PXD038198).

Growth hormone (GH) is a central regulator of β-cell proliferation, insulin secretion and sensitivity. Aim of this study was to investigate the effect of GH insensitivity on pancreatic β-cell histomorphology and consequences for metabolism in vivo. Pancreata from pigs with growth hormone receptor deficiency (GHR-KO, n = 12) were analyzed by unbiased quantitative stereology in comparison to wild-type controls (WT, n = 12) at 3 and 7–8.5 months of age. In vivo secretion capacity for insulin and glucose tolerance were assessed by intravenous glucose tolerance tests (ivGTTs) in GHR-KO (n = 3) and WT (n = 3) pigs of the respective age groups. Unbiased quantitative stereological analyses revealed a significant reduction in total β-cell volume (83

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN (acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE. In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygous frameshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

Objective: To gain mechanistic insights into adverse effects of maternal hyperglycemia on the liver of neonates, we performed a multi-omics analysis of liver tissue from piglets developed in genetically diabetic (mutant INS gene induced diabetes of youth; MIDY) or wild-type (WT) pigs. Methods: Proteome, metabolome and lipidome profiles of liver and clinical parameters of serum samples from 3-day-old WT piglets (n = 9) born to MIDY mothers (PHG) were compared with those of WT piglets (n = 10) born to normoglycemic mothers (PNG). Furthermore, protein-protein interaction network analysis was used to reveal highly interacting proteins that participate in the same molecular mechanisms and to relate these mechanisms with human pathology. Results: Hepatocytes of PHG displayed pronounced lipid droplet accumulation, although the abundances of central lipogenic enzymes such as fatty acid-synthase (FASN) were decreased. Additionally, circulating triglyceride (TG) levels were reduced as a trend. Serum levels of nonesterified free fatty acids (NEFA) were elevated in PHG, potentially stimulating hepatic gluconeogenesis. This is supported by elevated hepatic phosphoenolpyruvate carboxykinase (PCK1) and circulating alanine transaminase (ALT) levels. Even though targeted metabolomics showed strongly elevated phosphatidylcholine (PC) levels, the abundances of multiple key enzymes involved in major PC synthesis pathways - most prominently those from the Kennedy pathway - were paradoxically reduced in PHG liver. Conversely, enzymes involved in PC excretion and breakdown such as PC-specific translocase ATP-binding cassette 4 (ABCB4) and phospholipase A2 were increased in abundance. Conclusions: Our study indicates that maternal hyperglycemia without confounding obesity induces profound molecular changes in the liver of neonatal offspring. In particular, we found evidence for stimulated gluconeogenesis and hepatic lipid accumulation independent of de novo lipogenesis. Reduced levels of PC biosynthesis enzymes and increased levels of proteins involved in PC translocation or breakdown may represent counter-regulatory mechanisms to maternally elevated PC levels. Our comprehensive multi-omics dataset provides a valuable resource for future meta-analysis studies focusing on liver metabolism in newborns from diabetic mothers. & COPY; 2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Atrial fibrillation (AF) is the most prevalent arrhythmia, often caused by myocardial ischemia/infarction (MI). Men have a 1.5× higher prevalence of AF, whereas women show a higher risk for new onset AF after MI. However, the underlying mechanisms of how sex affects AF pathophysiology are largely unknown. In 72 pigs with/without ischemic heart failure (IHF) we investigated the impact of sex on ischemia-induced proarrhythmic atrial remodeling and the susceptibility for AF. Electrocardiogram (ECG) and electrophysiological studies were conducted to assess electrical remodeling; histological analyses were performed to assess atrial fibrosis in male and female pigs. IHF pigs of both sexes showed a significantly increased vulnerability for AF, but in male pigs more and longer episodes were observed. Unchanged conduction properties but enhanced left atrial fibrosis indicated structural rather than electrical remodeling underlying AF susceptibility. Sex differences were only observed in controls with female pigs showing an increased intrinsic heart rate, a prolonged QRS interval and a prolonged sinus node recovery time. In sum, susceptibility for AF is significantly increased both in male and female pigs with ischemic heart failure. Differences between males and females are moderate, including more and longer AF episodes in male pigs and sinus node dysfunction in female pigs.

Excessive dietary intake of fats and sugars (“Western diet”, WD) is one of the leading causes of obesity. The consumption of the microalga Arthrospira platensis (spirulina, Sp) is increasing due to its presumed health benefits. Both WD and Sp are also consumed by pregnant and breastfeeding women. This study investigated if gestating and lactating domestic pigs are an appropriate model for WD-induced metabolic disturbances similar to those observed in humans and if Sp supplementation may attenuate any of these adverse effects. Pigs were fed a WD high in fat, sugars, and cholesterol or a control diet. Half of the animals per diet group were supplemented with 20 g Sp per day. The WD did not increase body weight or adipose tissue accumulation but led to metabolic impairments such as higher cholesterol concentration in plasma, lower IGF1 plasma levels, and signs of hepatic damage compared to the control group. Spirulina supplementation could not reduce all the metabolic impairments observed in WD-fed animals. These findings indicate limited suitability of gestating and lactating domestic pigs as a model for WD but a certain potential of low-dose Sp supplementation to partially attenuate negative WD effects.

Diabetes mellitus is associated with an increased risk for arrhythmias such as atrial fibrillation (AF). The underlying mechanisms, however, are incompletely understood, but may include structural remodeling processes establishing a proarrhythmic atrial substrate.

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic INSC94Y transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favorable model to explore functional alterations of immune cells in an early stage of diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-diabetic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in INSC94Y tg pigs to polyclonal T cell mitogen phytohemagglutinin (PHA). Using label-free LC-MS/MS, a total of 3,487 proteins were quantified, and distinct changes in protein abundances in CD4+ T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic INSC94Y tg pigs, indicating an altered metabolic immune cell phenotype. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycemia.