Multi-omics analysis of diabetic pig lungs reveals molecular derangements underlying pulmonary complications of diabetes mellitus.

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 (WT) 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 (ECM) organization were significantly altered in abundance. Importantly, a lipoxygenase pathway was dysregulated as indicated by a 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 suffering from diabetes mellitus.

Structural and proteomic repercussions of growth hormone receptor deficiency on the pituitary gland: Lessons from a translational pig model.

Growth hormone receptor deficiency (GHRD) results in low serum insulin-like growth factor 1 (IGF1) and high, but non-functional serum growth hormone (GH) levels in human Laron syndrome (LS) patients and animal models. This study investigated the quantitative histomorphological and molecular alterations associated with GHRD. Pituitary glands from 6 months old growth hormone receptor deficient (GHR-KO) and control pigs were analyzed using a quantitative histomorphological approach in paraffin (9 GHR-KO [5 males, 4 females] vs. 11 controls [5 males, 6 females]), ultrathin sections tissue sections (3 male GHR-KO vs. 3 male controls) and label-free proteomics (4 GHR-KO vs. 4 control pigs [2 per sex]). GHR-KO pigs displayed reduced body weights (60% reduction in comparison to controls; p < .0001) and decreased pituitary volumes (54% reduction in comparison to controls; p < .0001). The volume proportion of the adenohypophysis did not differ in GHR-KO and control pituitaries (65% vs. 71%; p = .0506) and GHR-KO adenohypophyses displayed a reduced absolute volume but an unaltered volume density of somatotrophs in comparison to controls (21% vs. 18%; p = .3164). In GHR-KO pigs, somatotroph cells displayed a significantly reduced volume density of granules (23.5%) as compared to controls (67.7%; p < .0001). Holistic proteome analysis of adenohypophysis samples identified 4660 proteins, of which 592 were differentially abundant between the GHR-KO and control groups. In GHR-KO samples, the abundance of somatotropin precursor was decreased, whereas increased abundances of proteins involved in protein production, transport and endoplasmic reticulum (ER) stress were revealed. Increased protein production and secretion as well as significantly reduced proportion of GH-storing granules in somatotroph cells of the adenohypophysis without an increase in volume density of somatotroph cells in the adenohypophysis could explain elevated serum GH levels in GHR-KO pigs.

Proteomics- and Metabolomics-Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension.

Pulmonary vein stenosis (PVS) causes a rare type of pulmonary hypertension (PH) by impacting the flow and pressure within the pulmonary vasculature, resulting in endothelial dysfunction and metabolic changes. A prudent line of treatment in this type of PH would be targeted therapy to relieve the pressure and reverse the flow-related changes. We used a swine model in order to mimic PH after PVS using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to mimic the hemodynamic profile associated with PH and investigated the molecular alterations that provide an impetus for the development of PH. Our current study aimed to employ unbiased proteomic and metabolomic analyses on both the upper and lower lobes of the swine lung to identify regions with metabolic alterations. We detected changes in the upper lobes for the PVB animals mainly pertaining to fatty acid metabolism, reactive oxygen species (ROS) signaling and extracellular matrix (ECM) remodeling and small, albeit, significant changes in the lower lobes for purine metabolism.

Lysosomal TRPML1 regulates mitochondrial function in hepatocellular carcinoma cells.

Liver cancers, including hepatocellular carcinoma (HCC), are the second leading cause of cancer death worldwide, and novel therapeutic strategies are still highly needed. Recently, the endolysosomal cation channel TRPML1 (also known as MCOLN1) has gained focus in cancer research because it represents an interesting novel target. We utilized the recently developed isoform-selective TRPML1 activator ML1-SA1 and the CRISPR/Cas9 system to generate tools for overactivation and loss-of-function studies on TRPML1 in HCC. After verification of our tools, we investigated the role of TRPML1 in HCC by studying proliferation, apoptosis and proteomic alterations. Furthermore, we analyzed mitochondrial function in detail by performing confocal and transmission electron microscopy combined with SeahorseTM and Oroboros® functional analysis. We report that TRPML1 overactivation mediated by a novel, isoform-selective small-molecule activator induces apoptosis by impairing mitochondrial function in a Ca2+-dependent manner. Additionally, TRPML1 loss-of-function deregulates mitochondrial renewal, which leads to proliferation impairment. Thus, our study reveals a novel role for TRPML1 as regulator of mitochondrial function and its modulators as promising molecules for novel therapeutic options in HCC therapy.

Differential Effects of Insulin-Deficient Diabetes Mellitus on Visceral vs. Subcutaneous Adipose Tissue-Multi-omics Insights From the Munich MIDY Pig Model.

Adipose tissue (AT) is no longer considered to be responsible for energy storage only but is now recognized as a major endocrine organ that is distributed across different parts of the body and is actively involved in regulatory processes controlling energy homeostasis. Moreover, AT plays a crucial role in the development of metabolic disease such as diabetes. Recent evidence has shown that adipokines have the ability to regulate blood glucose levels and improve metabolic homeostasis. While AT has been studied extensively in the context of type 2 diabetes, less is known about how different AT types are affected by absolute insulin deficiency in type 1 or permanent neonatal diabetes mellitus. Here, we analyzed visceral and subcutaneous AT in a diabetic, insulin-deficient pig model (MIDY) and wild-type (WT) littermate controls by RNA sequencing and quantitative proteomics. Multi-omics analysis indicates a depot-specific dysregulation of crucial metabolic pathways in MIDY AT samples. We identified key proteins involved in glucose uptake and downstream signaling, lipogenesis, lipolysis and ß-oxidation to be differentially regulated between visceral and subcutaneous AT in response to insulin deficiency. Proteins related to glycogenolysis, pyruvate metabolism, TCA cycle and lipogenesis were increased in subcutaneous AT, whereas ß-oxidation-related proteins were increased in visceral AT from MIDY pigs, pointing at a regionally different metabolic adaptation to master energy stress arising from diminished glucose utilization in MIDY AT. Chronic, absolute insulin deficiency and hyperglycemia revealed fat depot-specific signatures using multi-omics analysis. The generated datasets are a valuable resource for further comparative and translational studies in clinical diabetes research.

Developmental Effects of (Pre-)Gestational Diabetes on Offspring: Systematic Screening Using Omics Approaches.

Worldwide, gestational diabetes affects 2-25% of pregnancies. Due to related disturbances of the maternal metabolism during the periconceptional period and pregnancy, children bear an increased risk for future diseases. It is well known that an aberrant intrauterine environment caused by elevated maternal glucose levels is related to elevated risks for increased birth weights and metabolic disorders in later life, such as obesity or type 2 diabetes. The complexity of disturbances induced by maternal diabetes, with multiple underlying mechanisms, makes early diagnosis or prevention a challenging task. Omics technologies allowing holistic quantification of several classes of molecules from biological fluids, cells, or tissues are powerful tools to systematically investigate the effects of maternal diabetes on the offspring in an unbiased manner. Differentially abundant molecules or distinct molecular profiles may serve as diagnostic biomarkers, which may also support the development of preventive and therapeutic strategies. In this review, we summarize key findings from state-of-the-art Omics studies addressing the impact of maternal diabetes on offspring health.

A scalable, clinically severe pig model for Duchenne muscular dystrophy.

Large-animal models for Duchenne muscular dystrophy (DMD) are crucial for the evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) exhibit molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/- carriers. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/- carriers resulted in an additional 114 DMDY/- piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance to human disease mechanisms. Importantly, DMDY/- pigs displayed progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction, at 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies.

Age-Related Alterations in the Testicular Proteome of a Non-Human Primate.

Aging of human testis and associated cellular changes is difficult to assess. Therefore, we used a translational, non-human primate model to get insights into underlying cellular and biochemical processes. Using proteomics and immunohistochemistry, we analyzed testicular tissue of young (age 2 to 3) and old (age 10 to 12) common marmosets (Callithrix jacchus). Using a mass spectrometry-based proteomics approach, we identified 63,124 peptides, which could be assigned to 5924 proteins. Among them, we found proteins specific for germ cells and somatic cells, such as Leydig and Sertoli cells. Quantitative analysis showed 31 differentially abundant proteins, of which 29 proteins were more abundant in older animals. An increased abundance of anti-proliferative proteins, among them CDKN2A, indicate reduced cell proliferation in old testes. Additionally, an increased abundance of several small leucine rich repeat proteoglycans and other extracellular matrix proteins was observed, which may be related to impaired cell migration and fibrotic events. Furthermore, an increased abundance of proteins with inhibitory roles in smooth muscle cell contraction like CNN1 indicates functional alterations in testicular peritubular cells and may mirror a reduced capacity of these cells to contract in old testes.

Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies.

BACKGROUND: Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step. METHODS: Heart weight and proteome profile of myocardium were investigated in GHR-KO and control pigs. GHR-KO mutations were introduced using CRISPR/Cas9 in an α1,3-galactosyltransferase (GGTA1)-deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple-modified (4GM) pigs. RESULTS: At age 6 months, GHR-KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin-like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi-modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. CONCLUSION: GHR-KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno-organ overgrowth in preclinical studies.

Filamin A Orchestrates Cytoskeletal Structure, Cell Migration and Stem Cell Characteristics in Human Seminoma TCam-2 Cells.

Filamins are large dimeric F-actin cross-linking proteins, crucial for the mechanosensitive properties of a number of cell types. Due to their interaction with a variety of different proteins, they exert important regulatory functions. However, in the human testis the role of filamins has been insufficiently explored. Immunohistochemical staining of human testis samples identified filamin A (FLNA) in spermatogonia and peritubular myoid cells. Investigation of different testicular tumor samples indicated that seminoma also express FLNA. Moreover, mass spectrometric analyses identified FLNA as one of the most abundant proteins in human seminoma TCam-2 cells. We therefore focused on FLNA in TCam-2 cells, and identified by co-immunoprecipitation LAD1, RUVBL1 and DAZAP1, in addition to several cytoskeletal proteins, as interactors of FLNA. To study the role of FLNA in TCam-2 cells, we generated FLNA-deficient cells using the CRISPR/Cas9 system. Loss of FLNA causes an irregular arrangement of the actin cytoskeleton and mechanical instability, impaired adhesive properties and disturbed migratory behavior. Furthermore, transcriptional activity of typical stem cell factors is increased in the absence of FLNA. In summary, our data suggest that FLNA is crucially involved in balancing stem cell characteristics and invasive properties in human seminoma cells and possibly human testicular germ cells.

Proteomic Insights into Senescence of Testicular Peritubular Cells from a Nonhuman Primate Model.

Age-related changes in the human testis may include morphological alterations, disturbed steroidogenesis, and impaired spermatogenesis. However, the specific impact of cell age remains poorly understood and difficult to assess. Testicular peritubular cells fulfill essential functions, including sperm transport, contributions to the spermatogonial stem cell niche, and paracrine interactions within the testis. To study their role in age-associated decline of testicular functions, we performed comprehensive proteome and secretome analyses of repeatedly passaged peritubular cells from Callithrix jacchus. This nonhuman primate model better reflects the human testicular biology than rodents and further gives access to young donors unavailable from humans. Among 5095 identified proteins, 583 were differentially abundant between samples with low and high passage numbers. The alterations indicate a reduced ability of senescent peritubular cells to contract and secrete proteins, as well as disturbances in nuclear factor (NF)-κB signaling and a reduced capacity to handle reactive oxygen species. Since this in vitro model may not exactly mirror all molecular aspects of in vivo aging, we investigated the proteomes and secretomes of testicular peritubular cells from young and old donors. Even though the age-related alterations at the protein level were less pronounced, we found evidence for impaired protein secretion, altered NF-κB signaling, and reduced contractility of these in vivo aged peritubular cells.

A decade of experience with genetically tailored pig models for diabetes and metabolic research.

The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes. Findings in rodent models are consequently often difficult to extrapolate to human clinical trials. To overcome this 'translational gap', we and other groups are developing porcine disease models. Pigs share many anatomical and physiological traits with humans and thus hold great promise as translational animal models. Importantly, the toolbox for genetic engineering of pigs is rapidly expanding. Human disease mechanisms and targets can therefore be reproduced in pigs on a molecular level, resulting in precise and predictive porcine (PPP) models. In this short review, we summarize our work on the development of genetically (pre)diabetic pig models and how they have been used to study disease mechanisms and test therapeutic strategies. This includes the generation of reporter pigs for studying beta-cell maturation and physiology. Furthermore, genetically engineered pigs are promising donors of pancreatic islets for xenotransplantation. In summary, genetically tailored pig models have become an important link in the chain of translational diabetes and metabolic research.

Progressive Proteome Changes in the Myocardium of a Pig Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is characterized by progressive muscle weakness. Even though DMD manifests first in skeletal muscle, heart failure is a major cause of death in late-stage DMD. To get insights into DMD-associated cardiomyopathy, we performed a proteome analysis of myocardium from a genetically engineered porcine DMD model resembling clinical and pathological hallmarks of human DMD. To capture DMD progression, samples from 2-day- and 3-month-old animals were analyzed. Dystrophin was absent in all DMD samples, and components of the dystrophin-associated protein complex were decreased, suggesting destabilization of the cardiomyocyte plasma membrane and impaired cellular signaling. Furthermore, abundance alterations of proteins known to be associated with human cardiomyopathy were observed. Compared with data from skeletal muscle, we found clear evidence that DMD progression in myocardium is not only slower than in skeletal muscle but also involves different biological and biochemical pathways.

The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†.

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC-MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell-cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

A translational cellular model for the study of peritubular cells of the testis.

Testicular peritubular cells (TPCs) are smooth muscle-like cells, which form a compartment surrounding the seminiferous tubules. Previous studies employing isolated human testicular peritubular cells (HTPCs) indicated that their roles in the testis go beyond sperm transport and include paracrine and immunological contributions. Peritubular cells from a non-human primate (MKTPCs), the common marmoset monkey, Callithrix jacchus, share a high degree of homology with HTPCs. However, like their human counterparts these cells age in vitro and replicative senescence limits in-depth functional or mechanistic studies. Therefore, a stable cellular model was established. MKTPCs of a young adult animal were immortalized by piggyBac transposition of human telomerase (hTERT), that is, without the expression of viral oncogenes. Immortalized MKTPCs (iMKTPCs) grew without discernable changes for more than 50 passages. An initial characterization revealed typical genes expressed by peritubular cells (androgen receptor (AR), smooth-muscle actin (ACTA2), calponin (CNN1)). A proteome analysis of the primary MKTPCs and the derived immortalized cell line confirmed that the cells almost completely retained their phenotype. To test whether they respond in a similar way as HTPCs, iMKTPCs were challenged with forskolin (FSK) and ATP. As HTPCs, they showed increased expression level of the StAR protein (StAR) after FSK stimulation, indicating steroidogenic capacity. ATP increased the expression of pro-inflammatory factors (e.g. IL1B; CCL7), as it is the case in HTPCs. Finally, we confirmed that iMKTPCs can efficiently be transfected. Therefore, they represent a highly relevant translational model, which allows mechanistic studies for further exploration of the roles of testicular peritubular cells.

Genetic merit for fertility alters the bovine uterine luminal fluid proteome†.

Over the last decades, fertility of dairy cows has declined due to selection strategies focusing on milk yield. To study the effect of genetic merit for fertility on the proteome of the bovine uterine luminal fluid, Holstein heifers with low- and two groups of heifers with high-fertility index (high-fertility Holstein and Montbéliarde) were investigated. To focus on the maternal effect, heifers from all groups were synchronized and received on Day 7 high-quality embryos. Uterine luminal fluid from Day 19 pregnant heifers was analyzed in a holistic proteomic approach using nano-LC-MS/MS analysis combined with a label-free quantification approach. In total, 1737 proteins were identified, of which 597 differed significantly in abundance between the three groups. The vast majority of proteome differences was found comparing both high-fertility groups to the low-fertility Holstein group, showing that the genetic predisposition for fertility is prevalent regarding the uterine luminal fluid proteome. Evaluation of this dataset using bioinformatic tools revealed an assignment of higher abundant proteins in low-fertility Holstein to several metabolic processes, such as vitamin metabolic process, which comprises folate receptor alpha (FOLR1) and retinol-binding protein, indicating an involvement of disturbed metabolic processes in decreased fertility. Moreover, immune system-related proteins - lactotransferrin and chromogranin A - were enriched in low-fertility cows together with interferon tau 3 h and interferon tau-2. Our results indicate that the genetic merit for fertility leads to substantial quantitative differences at the level of proteins in uterine fluid of pregnant animals, thus altering the microenvironment for the early conceptus.

Detection of collagens by multispectral optoacoustic tomography as an imaging biomarker for Duchenne muscular dystrophy.

Biomarkers for monitoring of disease progression and response to therapy are lacking for muscle diseases such as Duchenne muscular dystrophy. Noninvasive in vivo molecular imaging with multispectral optoacoustic tomography (MSOT) uses pulsed laser light to induce acoustic pressure waves, enabling the visualization of endogenous chromophores. Here we describe an application of MSOT, in which illumination in the near- and extended near-infrared ranges from 680-1,100 nm enables the visualization and quantification of collagen content. We first demonstrated the feasibility of this approach to noninvasive quantification of tissue fibrosis in longitudinal studies in a large-animal Duchenne muscular dystrophy model in pigs, and then applied this approach to pediatric patients. MSOT-derived collagen content measurements in skeletal muscle were highly correlated to the functional status of the patients and provided additional information on molecular features as compared to magnetic resonance imaging. This study highlights the potential of MSOT imaging as a noninvasive, age-independent biomarker for the implementation and monitoring of newly developed therapies in muscular diseases.

Insights into replicative senescence of human testicular peritubular cells.

There is evidence for an age-related decline in male reproductive functions, yet how the human testis may age is not understood. Human testicular peritubular cells (HTPCs) transport sperm, contribute to the spermatogonial stem cell (SSC) niche and immune surveillance, and can be isolated and studied in vitro. Consequences of replicative senescence of HTPCs were evaluated to gain partial insights into human testicular aging. To this end, early and advanced HTPC passages, in which replicative senescence was indicated by increased cell size, altered nuclear morphology, enhanced ß-galactosidase activity, telomere attrition and reduced mitochondrial DNA (mtDNA), were compared. These alterations are typical for senescent cells, in general. To examine HTPC-specific changes, focused ion beam scanning electron microscopy (FIB/SEM) tomography was employed, which revealed a reduced mitochondrial network and an increased lysosome population. The results coincide with the data of a parallel proteomic analysis and indicate deranged proteostasis. The mRNA levels of typical contractility markers and growth factors, important for the SSC niche, were not significantly altered. A secretome analysis identified, however, elevated levels of macrophage migration inhibitory factor (MIF) and dipeptidyl peptidase 4 (DPP4), which may play a role in spermatogenesis. Testicular DPP4 may further represent a possible drug target.

Multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus.

OBJECTIVE: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. METHODS: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics. RESULTS: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. CONCLUSIONS: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets.

The Munich MIDY Pig Biobank - A unique resource for studying organ crosstalk in diabetes.

OBJECTIVE: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. METHODS: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. RESULTS: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. CONCLUSIONS: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.