Background: Animal models of non-alcoholic steatohepatitis (NASH) are important tools in preclinical research and drug discovery. Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mice represent a model of fibrosing NASH. The present study directly assessed the clinical translatability of the model by head-to-head comparison of liver biopsy histological and transcriptome changes in GAN DIO-NASH mouse and human NASH patients. Methods: C57Bl/6 J mice were fed chow or the GAN diet rich in saturated fat (40%), fructose (22%) and cholesterol (2%) for ≥38 weeks. Metabolic parameters as well as plasma and liver biomarkers were assessed. Liver biopsy histology and transcriptome signatures were compared to samples from human lean individuals and patients diagnosed with NASH. Results: Liver lesions in GAN DIO-NASH mice showed similar morphological characteristics compared to the NASH patient validation set, including macrosteatosis, lobular inflammation, hepatocyte ballooning degeneration and periportal/perisinusoidal fibrosis. Histomorphometric analysis indicated comparable increases in markers of hepatic lipid accumulation, inflammation and collagen deposition in GAN DIO-NASH mice and NASH patient samples. Liver biopsies from GAN DIO-NASH mice and NASH patients showed comparable dynamics in several gene expression pathways involved in NASH pathogenesis. Consistent with the clinical features of NASH, GAN DIO-NASH mice demonstrated key components of the metabolic syndrome, including obesity and impaired glucose tolerance. Conclusions: The GAN DIO-NASH mouse model demonstrates good clinical translatability with respect to the histopathological, transcriptional and metabolic aspects of the human disease, highlighting the suitability of the GAN DIO-NASH mouse model for identifying therapeutic targets and characterizing novel drug therapies for NASH.

to improve the understanding of the complex biological processes underlying the development of non-alcoholic steatohepatitis (nASH), a multi-omics approach combining bulk RnA-sequencing based transcriptomics, quantitative proteomics and single-cell RnA-sequencing was used to characterize tissue biopsies from histologically validated diet-induced obese (Dio) nASH mice compared to chow-fed controls. Bulk RnA-sequencing and proteomics showed a clear distinction between phenotypes and a good correspondence between mRnA and protein level regulations, apart from specific regulatory events discovered by each technology. Transcriptomics-based gene set enrichment analysis revealed changes associated with key clinical manifestations of nASH, including impaired lipid metabolism, increased extracellular matrix formation/remodeling and pro-inflammatory responses, whereas proteomics-based gene set enrichment analysis pinpointed metabolic pathway perturbations. Integration with single-cell RNA-sequencing data identified key regulated cell types involved in development of nASH demonstrating the cellular heterogeneity and complexity of nASH pathogenesis. Non-alcoholic fatty liver disease (NALFD) comprises a wide spectrum of liver diseases ranging from typically benign steatosis to non-alcoholic steatohepatitis (NASH), with or without fibrosis, that can progress into cirrhosis, hepatocellular carcinoma and ultimately end-stage liver disease 1-3. The development of NASH is driven by complex and dynamic molecular mechanisms, implicating multiple parallel signalling pathways. However, the interplay between these different clinical and molecular manifestations linked to progression of NAFLD into NASH is not fully understood. Currently, no pharmacological therapies for NASH exist, however lifestyle modifications have shown to be efficacious for NASH resolution 4. There are no early diagnostic endpoints known, and since hepatic steatosis and fibrosis can present itself as asymptomatic there is an unmet need to better understand the etiology and pathogenesis of NASH. Accordingly, several rodent models mimicking pathological features of NASH have been developed to accommodate this 5. Due to the distinct hepatic features of NASH, histological techniques based on qualitative scoring systems 6,7 and quantitative image analysis have been developed for research applications. Omics-based strategies have been instrumental for hypothesis-free analysis of molecular changes in NASH. For example, genome-wide association studies have identified several loci with variants showing increased risk of NAFLD and NASH development 8-11 or protection from more aggressive liver pathologies 12. Furthermore, transcriptomics has been successful in identifying novel regulatory mechanism in NASH pathogenesis 13-17. Despite the value of transcriptomics, these approaches will not always reflect the actual abundance at the protein level of a given the specific gene product in the cell 18 or extracellular space/surrounding body fluids 19. Quantitative proteomics has su...

The current understanding of molecular mechanisms driving diabetic kidney disease (DKD) is limited, partly due to the complex structure of the kidney. To identify genes and signalling pathways involved in the progression of DKD, we compared kidney cortical vs. glomerular transcriptome profiles in uninephrectomized (UNx) db/db mouse models of early-stage (UNx only) and advanced (UNx plus AAV-mediated renin overexpression, UNx-Renin) DKD using RNA sequencing (RNAseq). Compared to normoglycemic db/m mice, db/db UNx and db/db UNx-Renin mice showed marked changes in kidney cortical and glomerular gene expression profiles. UNx-Renin mice displayed more marked perturbations in gene components associated with activation of the immune system and enhanced extracellular matrix remodelling, supporting histological hallmarks of progressive DKD in this model. Single-nucleus RNAseq enabled linking transcriptome profiles to specific kidney cell types. In conclusion, integration of RNAseq at the cortical, glomerular and single-nucleus level provides enhanced resolution of molecular signalling pathways associated with disease progression in preclinical models of DKD, and may thus be advantageous for identifying novel therapeutic targets in DKD.

Non-invasive biomarkers of non-alcoholic fatty liver disease (NAFLD) supporting diagnosis and monitoring disease progression are urgently needed. The present study aimed to establish a bioinformatics pipeline capable of defining and validating NAFLD biomarker candidates based on paired hepatic global gene expression and plasma bioanalysis from individuals representing different stages of histologically confirmed NAFLD (no/mild, moderate, more advanced NAFLD). Liver secretome gene signatures were generated in a patient cohort of 26 severely obese individuals with the majority having no or mild fibrosis. To this end, global gene expression changes were compared between individuals with no/mild NAFLD and moderate/advanced NAFLD with subsequent filtering for candidate gene products with liver-selective expression and secretion. Four candidate genes, including LPA (lipoprotein A), IGFBP-1 (insulin-like growth factor-binding protein 1), SERPINF2 (serpin family F member 2) and MAT1A (methionine adenosyltransferase 1A), were differentially expressed in moderate/advanced NAFLD, which was confirmed in three independent RNA sequencing datasets from large, publicly available NAFLD studies. The corresponding gene products were quantified in plasma samples but could not discriminate among different grades of NAFLD based on NAFLD activity score. Conclusion: We demonstrate a novel approach based on the liver transcriptome allowing for identification of secreted hepatic gene products as potential circulating diagnostic biomarkers of NAFLD. Using this approach in larger NAFLD patient cohorts may yield potential circulating biomarkers for NAFLD severity.

The hypocretin/orexin system regulates arousal through central nervous system mechanisms and plays an important role in sleep, wakefulness, and energy homeostasis. It is unclear whether hypocretin peptides are also present in blood due to difficulties in measuring reliable and reproducible levels of the peptides in blood samples. Lack of hypocretin signaling causes the sleep disorder Narcolepsy Type 1, and low concentration of cerebrospinal fluid hypocretin-1/orexin-A peptide is a hallmark of the disease. This measurement has high diagnostic value, but performing a lumbar puncture is not without discomfort and possible complications for the patient. A blood-based test to assess hypocretin-1 deficiency would therefore be of obvious benefit. We here demonstrate that heating plasma or serum samples to 65 °C for 30 min at pH 8 significantly increases hypocretin-1 immunoreactivity enabling stable and reproducible measurement of hypocretin-1 in blood samples. Specificity of the signal was verified by high-performance liquid chromatography and by measuring blood samples from mice lacking hypocretin. Unspecific background signal in the assay was high. Using our method, we show that hypocretin-1 immunoreactivity in blood samples from Narcolepsy Type 1 patients does not differ from the levels detected in control samples. The data presented here suggest that hypocretin-1 is present in the blood stream in the low picogram/mL range and that peripheral hypocretin-1 concentrations are unchanged in Narcolepsy Type 1. The hypocretin/orexin system in the brain regulates sleep and wakefulness. Aegidius et al. have developed a simple test to measure hypocretin-1 peptide in blood samples, and show that peripheral hypocretin-1 concentrations are unchanged in Narcolepsy Type 1 despite being absent in the brains of these patients.