Birgit Rathkolb

Aims/hypothesis: Offspring of mothers with diabetes are at increased risk of developing type 2 diabetes, and this predisposition is established during fetal life. The aim of this study was to define the functional, morphological and molecular alterations in neonatal pancreatic islets associated with maternal hyperglycaemia, with a particular focus on beta cells, an area that remains poorly understood. Given its similarity to humans in endocrine pancreas development, the pig was used as a translational animal model. Methods: Using genetically diabetic and beta cell reporter pig models, we performed functional assessments of glucose-stimulated insulin secretion in vivo and in situ using fresh pancreatic tissue slices from neonatal piglets born to hyperglycaemic mothers (PHGs) or normoglycaemic mothers (PNGs). Morphological and molecular profiling, including proteomics and single-cell RNA-seq, were performed to elucidate molecular changes reflecting the developmental impact of maternal diabetes on offspring metabolic health, with a particular focus on neonatal beta cells. Results: PHGs were born hyperglycaemic, hyperinsulinaemic and insulin resistant, exhibiting markedly delayed glucose and insulin response curves following oral glucose administration, with more pronounced effects in male piglets. Elevated basal insulin secretion and an almost blunted glucose responsiveness of pancreatic tissue slices were associated with increased insulin and somatostatin content, expansion of beta (+35%) and delta (+47%) cell populations, and organisation into larger islet clusters. Neonatal PHG islets exhibited a molecular profile consistent with intrinsic beta cell dysfunction that partially overlapped with type 2 diabetes (e.g. RBP4 upregulation), together with alterations in glucose sensing (near absence of GLUT2), nutrient metabolism resembling congenital hyperinsulinism ( HADH downregulation), and cytosolic Ca ² ⁺ homeostasis (near absence of the Ca ² ⁺ buffer calbindin D28K). In addition to an ATF4-dominated stress signature, a shift towards a distinct PHG beta cell subcluster characterised by low expression of ribosomal genes was observed, with an adaptive early-response-gene-mediated transcriptional reactivity. In contrast to the Gαs–adenylyl cyclase–cAMP–PKA signalling axis that dominated in PNG beta cells, neonatal PHG beta cells displayed distinct alterations in Gαq–PLC–PKCα signalling components, the mTORC2 target PKCα, and CREB1, suggesting a restructured signalling architecture. Conclusions/interpretation: Our findings reveal altered islet modelling with a unique beta cell dysfunction profile that partially overlaps with, but differs from, that of type 2 diabetes. A shift towards a beta cell subcluster exhibiting an adaptive early-response-gene-mediated transcriptional reactivity and an ATF4-associated transcriptional stress signature provides insight into molecular alterations associated with fetal exposure to maternal diabetes and may contribute to the increased risk of metabolic disease in offspring.

Near‐infrared (NIR) cyanine dyes demonstrate considerable promise in medical applications, notably in cancer diagnosis, due to their favourable fluorescence properties and biocompatibility. However, these dyes generally have low photostability and tend to aggregate in aqueous solution, which can constrain their usefulness for prolonged in vivo imaging and phototherapy applications. Here, we report the synthesis of a selenium‐integrated NIR cyanine dye, Se‐Cyn, designed to modulate its electronic and photophysical properties. Se‐Cyn demonstrates high photostability, molar absorptivity, photothermal conversion efficiency (PCE), and sustained optoacoustic (OptA) contrast under prolonged irradiation. Se‐Cyn nanoparticles (NPs) outperform the indocyanine green (ICG) NPs, exhibiting PCE of 59% and a 1.36‐fold higher OptA generation efficiency. Additionally, nano‐formulated Se‐Cyn NPs show excellent aqueous stability and improved bioavailability, supporting its suitability for biological applications. Furthermore, Se‐Cyn NPs accumulate in tumors following systemic administration, enabling effective OptA tumor imaging. Together with their strong photodynamic and photothermal properties, these NPs show considerable potential for cancer theranostics.

Histone variants with metabolite-binding macrodomains provide a poorly understood link between chromatin composition and metabolism. To address their contribution to physiological health, we generated and analyzed mice individually lacking the histone variants macroH2A1.1, macroH2A1.2, or macroH2A2. We identified several histopathologic changes in the kidney as isoform-specific phenotype of complete macroH2A1.1 loss affecting male and female animals. Kidney alterations were barely associated with organ-intrinsic gene expression changes but strongly correlated with a systemic shift in nutrient metabolization and alterations in NAD ⁺ (nicotinamide adenine dinucleotide, oxidized form) metabolism. Reduced lipid oxidation and increased glycolysis were found in male and female mice lacking macroH2A1.1 but not macroH2A1.2 or macroH2A2. Male macroH2A1.1-deficient mice also had better glucose tolerance and altered hepatic gene expression. Replacing chow by ketogenic diet overrode the macroH2A1.1-dependent metabolic phenotype and prevented kidney abnormalities. Together, our results indicate that macroH2A1.1 controls nutrient metabolization and links macroH2A1.1 levels to secondary changes in the kidney.

The lncRNA Crossfirre was identified as an imprinted X-linked gene, and is transcribed antisense to the trans-acting lncRNA Firre. The Firre locus forms an inactive-X-specific interaction with Dxz4, both loci providing the platform for the largest conserved chromatin structures. Here, we characterize the epigenetic profile of these loci, revealing them as the most female-specific accessible regions genome-wide. To address their in vivo role, we perform one of the largest X-linked knockout studies by deleting Crossfirre, Firre, and Dxz4 individually and in combination. Despite their distinct epigenetic features observed on the X chromosome, our allele-specific analysis uncovers these loci as dispensable for imprinted and random X chromosome inactivation. However, we provide evidence that Crossfirre affects autosomal gene regulation but only in combination with Firre. To shed light on the functional role of these sex-specific loci, we perform an extensive standardized phenotyping pipeline and uncover diverse knockout and sex-specific phenotypes. Collectively, our study provides the foundation for exploring the intricate interplay of conserved X-linked loci in vivo.

Thyroid hormone (TH) effects are mediated through TH receptors (TRs) TRα1, TRβ1, and TRβ2. The TRs bind to the DNA and regulate expression of TH target genes (canonical signaling). In addition, they mediate activation of signaling pathways (noncanonical signaling). Whether noncanonical TR action contributes to the spectrum of TH effects is largely unknown. Aim of this study was to attribute physiological effects to the TR isoforms and their canonical and noncanonical signaling. We conducted multi-parameter phenotyping in male and female TR knockout mice (TRα^KO, TRβ^KO), mice with disrupted canonical signaling due to mutations in the TR DNA-binding domain (TRα^GS, TRβ^GS) and their wild-type littermates. Perturbations in senses, especially hearing (mainly TRβ with a lesser impact of TRα), visual acuity, retinal thickness (TRα and TRβ) and in muscle metabolism (TRα) highlighted the role of canonical TR action. Strikingly, selective abrogation of canonical TR action often had little phenotypic consequence, suggesting that noncanonical TR action sufficed to maintain the wild-type phenotype for specific effects. For instance, macrocytic anemia, reduced retinal vascularization or increased anxiety related behavior were only observed in TRα^KO, but not TRαGS mice. Noncanonical TRα action improved energy utilization and prevented hyperphagia observed in female TRα^KO mice. In summary, by examining the phenotypes of TRα and TRβ knockout models alongside their DNA-binding-deficient mutants and wild-type counterparts, we could establish that the noncanonical actions of TRα and TRβ play a crucial role in modulating sensory, behavioral, and metabolic functions and, thus, contribute to the spectrum of physiological TH effects.

Thyroid hormone (TH) effects are mediated through TH receptors (TRs) TRα1, TRβ1, and TRβ2. The TRs bind to thyroid hormone responsive elements on the DNA and regulate expression of TH target genes as ligand dependent transcription factors (canonical signaling). In addition, the TRs α and β mediate activation of signaling pathways, e.g. the PI3K/AKT and MAPK/ERK pathways (noncanonical signaling). Whether such DNA-binding independent TR action contributes to the spectrum of physiological TH effects is largely unknown. The aim of this study was to attribute physiological effects to the two TR isoforms α and β and their canonical and noncanonical signaling. We conducted multi-parameter phenotyping in male and female TR knockout mice (TRα ^KO , TRβ ^KO ), mice with disrupted canonical signaling due to a mutation in the TR DNA-binding domain (TRα ^GS , TRβ ^GS ) and their respective wild-type littermates. Perturbations in senses, especially hearing (mainly TRβ with a lesser impact of TRα), visual acuity and retinal thickness (TRα and TRβ), in muscle metabolism (TRα) and in heart rate (TRα) highlighted the role of canonical TR action. Strikingly, selective abrogation of canonical TR action often had little to no phenotypic consequence, suggesting that noncanonical TR action sufficed to maintain the wild-type phenotype for specific effects. For instance, macrocytic anemia, reduced retinal vascularization or increased anxiety related behavior were only observed in TRα ^KO , but not TRα ^GS mice. Noncanonical TRα action increased the efficiency of energy utilization and prevented hyperphagia observed in TRα ^KO mice. In summary, by examining the phenotypes of TRα and TRβ knockout models alongside their DNA-binding-deficient GS mutants and wildtype counterparts, we could establish that the independent noncanonical actions of TRα and TRβ play a crucial role in modulating sensory, behavioral, and metabolic functions. This comparison underscores the significance of the TRs in orchestrating a spectrum of physiological processes beyond their traditional genomic pathways.

Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria, present unique challenges to energetic homeostasis by disrupting energy-producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut)–type methylmalonic aciduria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared with littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator–activated receptor and Fgf21-controlled pathways. Together, these shed light on the mechanisms and adaptations behind energy imbalance in methylmalonic aciduria and provide insight into metabolic responses to chronic energy shortage, which may have important implications for disease understanding and patient management.

Rare diseases (RDs) are a challenge for medicine due to their heterogeneous clinical manifestations and low prevalence. There is a lack of specific treatments and only a few hundred of the approximately 7,000 RDs have an approved regime. Rapid technological development in genome sequencing enables the mass identification of potential candidates that in their mutated form could trigger diseases but are often not confirmed to be causal. Knockout (KO) mouse models are essential to understand the causality of genes by allowing highly standardized research into the pathogenesis of diseases. The German Mouse Clinic (GMC) is one of the pioneers in mouse research and successfully uses (preclinical) data obtained from single-gene KO mutants for research into monogenic RDs. As part of the International Mouse Phenotyping Consortium (IMPC) and INFRAFRONTIER, the pan-European consortium for modeling human diseases, the GMC expands these preclinical data toward global collaborative approaches with researchers, clinicians, and patient groups.Here, we highlight proprietary genes that when deleted mimic clinical phenotypes associated with known RD targets (Nacc1, Bach2, Klotho alpha). We focus on recognized RD genes with no pre-existing KO mouse models (Kansl1l, Acsf3, Pcdhgb2, Rabgap1, Cox7a2) which highlight novel phenotypes capable of optimizing clinical diagnosis. In addition, we present genes with intriguing phenotypic data (Zdhhc5, Wsb2) that are not presently associated with known human RDs.This report provides comprehensive evidence for genes that when deleted cause differences in the KO mouse across multiple organs, providing a huge translational potential for further understanding monogenic RDs and their clinical spectrum. Genetic KO studies in mice are valuable to further explore the underlying physiological mechanisms and their overall therapeutic potential.