Hepatoprotective effects of the long-acting fibroblast growth factor 21 analog PF-05231023 in the GAN diet-induced obese and biopsy-confirmed mouse model of nonalcoholic steatohepatitis.

Fibroblast growth factor 21 (FGF21) plays a key role in hepatic lipid metabolism and long-acting FGF21 analogs have emerged as promising drug candidates for the treatment of nonalcoholic steatohepatitis (NASH). It remains to characterize this drug class in translational animal models that recapitulate the etiology and hallmarks of human disease. To this end, we evaluated the long-acting FGF21 analog PF-05231023 in the GAN (Gubra Amylin NASH) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. Male C57BL/6J mice were fed the GAN diet high in fat, fructose, and cholesterol for 34 wk before the start of the study. GAN DIO-NASH mice with biopsy-confirmed NAFLD Activity Score (NAS ≥5) and fibrosis (stage ≥F1) were biweekly administered with PF-05231023 (10 mg/kg sc) or vehicle (sc) for 12 wk. Vehicle-dosed chow-fed C57BL/6J mice served as healthy controls. Pre-to-post liver biopsy histopathological scoring was performed for within-subject evaluation of NAFLD Activity Score (NAS) and fibrosis stage. Terminal endpoints included quantitative liver histology and transcriptome signatures as well as blood and liver biochemistry. PF-05231023 significantly reduced body weight, hepatomegaly, plasma transaminases, and plasma/liver lipids in GAN DIO-NASH mice. Notably, PF-05231023 reduced both NAS (≥2-point improvement) and fibrosis stage (1-point improvement). Improvements in NASH and fibrosis severity were supported by reduced quantitative histological markers of steatosis, inflammation, and fibrogenesis as well as improvements in disease-associated liver transcriptome signatures. In conclusion, PF-05231023 reduces NASH and fibrosis severity in a translational biopsy-confirmed mouse model of NASH, supporting development of FGF21 analogs for the treatment of NASH.NEW & NOTEWORTHY It is unclear if long-acting FGF21 analogs have antifibrotic efficacy in NASH. We therefore profiled the clinically relevant FGF21 analog PF-05231023 in a translational diet-induced obese and biopsy-confirmed mouse model of NASH. We found PF-05231023 to exert hepatoprotective effects as indicated by notable improvements in plasma markers and histological hallmarks of NASH, including improved fibrosis stage. Collectively, the present study supports the continued exploration of long-acting FGF21 analogs for the treatment of NASH and other fibrotic diseases.

A liver secretome gene signature-based approach for determining circulating biomarkers of NAFLD severity.

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.

Lipidated PrRP31 metabolites are long acting dual GPR10 and NPFF2 receptor agonists with potent body weight lowering effect.

Prolactin-releasing peptide (PrRP) is an endogenous neuropeptide involved in appetite regulation and energy homeostasis. PrRP binds with high affinity to G-protein coupled receptor 10 (GPR10) and with lesser activity towards the neuropeptide FF receptor type 2 (NPFF2R). The present study aimed to develop long-acting PrRP31 analogues with potent anti-obesity efficacy. A comprehensive series of C18 lipidated PrRP31 analogues was characterized in vitro and analogues with various GPR10 and NPFF2R activity profiles were profiled for bioavailability and metabolic effects following subcutaneous administration in diet-induced obese (DIO) mice. PrRP31 analogues acylated with a C18 lipid chain carrying a terminal acid (C18 diacid) were potent GPR10-selective agonists and weight-neutral in DIO mice. In contrast, acylation with aliphatic C18 lipid chain (C18) resulted in dual GPR10-NPFF2R co-agonists that suppressed food intake and promoted a robust weight loss in DIO mice, which was sustained for at least one week after last dosing. Rapid in vivo degradation of C18 PrRP31 analogues gave rise to circulating lipidated PrRP metabolites maintaining dual GPR10-NPFF2R agonist profile and long-acting anti-obesity efficacy in DIO mice. Combined GPR10 and NPFF2R activation may therefore be a critical mechanism for obtaining robust anti-obesity efficacy of PrRP31 analogues.

Hepatoprotective effects of semaglutide, lanifibranor and dietary intervention in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH.

Non-alcoholic steatohepatitis (NASH) has emerged as a major challenge for public health because of high global prevalence and lack of evidence-based therapies. Most animal models of NASH lack sufficient validation regarding disease progression and pharmacological treatment. The Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mouse demonstrate clinical translatability with respect to disease etiology and hallmarks of NASH. This study aimed to evaluate disease progression and responsiveness to clinically effective interventions in GAN DIO-NASH mice. Disease phenotyping was performed in male C57BL/6J mice fed the GAN diet high in fat, fructose, and cholesterol for 28-88 weeks. GAN DIO-NASH mice with biopsy-confirmed NASH and fibrosis received low-caloric dietary intervention, semaglutide (30 nmol/kg/day, s.c.) or lanifibranor (30 mg/kg/day, p.o.) for 8 and 12 weeks, respectively. Within-subject change in nonalcoholic fatty liver disease (NAFLD) Activity Score (NAS) and fibrosis stage was evaluated using automated deep learning-based image analysis. GAN DIO-NASH mice showed clear and reproducible progression in NASH, fibrosis stage, and tumor burden with high incidence of hepatocellular carcinoma. Consistent with clinical trial outcomes, semaglutide and lanifibranor improved NAS, whereas only lanifibranor induced regression in the fibrosis stage. Dietary intervention also demonstrated substantial benefits on metabolic outcomes and liver histology. Differential therapeutic efficacy of semaglutide, lanifibranor, and dietary intervention was supported by quantitative histology, RNA sequencing, and blood/liver biochemistry. In conclusion, the GAN DIO-NASH mouse model recapitulates various histological stages of NASH and faithfully reproduces histological efficacy profiles of compounds in advanced clinical development for NASH. Collectively, these features highlight the utility of GAN DIO-NASH mice in preclinical drug development.

Integrative transcriptomic profiling of a mouse model of hypertension-accelerated diabetic kidney disease.

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 versus glomerular transcriptome profiles in uninephrectomized (UNx) db/db mouse models of early-stage (UNx only) and advanced [UNxplus adeno-associated virus-mediated renin-1 overexpression (UNx-Renin)] DKD using RNAseq. Compared to normoglycemic db/m mice, db/db UNx and db/db UNx-Renin mice showed marked changes in their kidney cortical and glomerular gene expression profiles. UNx-Renin mice displayed more marked perturbations in gene components associated with the activation of the immune system and enhanced extracellular matrix remodelling, supporting histological hallmarks of progressive DKD in this model. Single-nucleus RNAseq enabled the linking of transcriptome profiles to specific kidney cell types. In conclusion, integration of RNAseq at the cortical, glomerular and single-nucleus level provides an 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.

Therapeutic effects of lisinopril and empagliflozin in a mouse model of hypertension-accelerated diabetic kidney disease.

Hypertension is a critical comorbidity for progression of diabetic kidney disease (DKD). To facilitate the development of novel therapeutic interventions with the potential to control disease progression, there is a need to establish translational animal models that predict treatment effects in human DKD. The present study aimed to characterize renal disease and outcomes of standard of medical care in a model of advanced DKD facilitated by adeno-associated virus (AAV)-mediated renin overexpression in uninephrectomized (UNx) db/db mice. Five weeks after single AAV administration and 4 wk after UNx, female db/db UNx-ReninAAV mice received (PO, QD) vehicle, lisinopril (40 mg/kg), empagliflozin (20 mg/kg), or combination treatment for 12 wk (n = 17 mice/group). Untreated db/+ mice (n = 8) and vehicle-dosed db/db UNx-LacZAAV mice (n = 17) served as controls. End points included plasma, urine, and histomorphometric markers of kidney disease. Total glomerular numbers and individual glomerular volume were evaluated by whole kidney three-dimensional imaging analysis. db/db UNx-ReninAAV mice developed hallmarks of progressive DKD characterized by severe albuminuria, advanced glomerulosclerosis, and glomerular hypertrophy. Lisinopril significantly improved albuminuria, glomerulosclerosis, tubulointerstitial injury, and inflammation. Although empagliflozin alone had no therapeutic effect on renal endpoints, lisinopril and empagliflozin exerted synergistic effects on renal histological outcomes. In conclusion, the db/db UNx-ReninAAV mouse demonstrates good clinical translatability with respect to physiological and histological hallmarks of progressive DKD. The efficacy of standard of care to control hypertension and hyperglycemia provides a proof of concept for testing novel drug therapies in the model.NEW & NOTEWORTHY Translational animal models of diabetic kidney disease (DKD) are important tools in preclinical research and drug discovery. Here, we show that the standard of care to control hypertension (lisinopril) and hyperglycemia (empagliflozin) improves physiological and histopathological hallmarks of kidney disease in a mouse model of hypertension-accelerated progressive DKD. The findings substantiate hypertension and type 2 diabetes as essential factors in driving DKD progression and provide a proof of concept for probing novel drugs for potential nephroprotective efficacy in this model.

Whole-brain mapping of amylin-induced neuronal activity in receptor activity-modifying protein 1/3 knockout mice.

The pancreatic hormone amylin plays a central role in regulating energy homeostasis and glycaemic control by stimulating satiation and reducing food reward, making amylin receptor agonists attractive for the treatment of metabolic diseases. Amylin receptors consist of heterodimerized complexes of the calcitonin receptor and receptor-activity modifying proteins subtype 1-3 (RAMP1-3). Neuronal activation in response to amylin dosing has been well characterized, but only in selected regions expressing high levels of RAMPs. The current study identifies global brain-wide changes in response to amylin and by comparing wild type and RAMP1/3 knockout mice reveals the importance of RAMP1/3 in mediating this response. Amylin dosing resulted in neuronal activation as measured by an increase in c-Fos labelled cells in 20 brain regions, altogether making up the circuitry of neuronal appetite regulation (e.g., area postrema (AP), nucleus of the solitary tract (NTS), parabrachial nucleus (PB), and central amygdala (CEA)). c-Fos response was also detected in distinct nuclei across the brain that typically have not been linked with amylin signalling. In RAMP1/3 knockout amylin induced low-level neuronal activation in seven regions, including the AP, NTS and PB, indicating the existence of RAMP1/3-independent mechanisms of amylin response. Under basal conditions RAMP1/3 knockout mice show reduced neuronal activity in the hippocampal formation as well as reduced hippocampal volume, suggesting a role for RAMP1/3 in hippocampal physiology and maintenance. Altogether these data provide a global map of amylin response in the mouse brain and establishes the significance of RAMP1/3 receptors in relaying this response.

Characterization of combined linagliptin and Y2R agonist treatment in diet-induced obese mice.

Dipeptidyl peptidase IV (DPP-IV) inhibitors improve glycemic control by prolonging the action of glucagon-like peptide-1 (GLP-1). In contrast to GLP-1 analogues, DPP-IV inhibitors are weight-neutral. DPP-IV cleavage of PYY and NPY gives rise to PYY3-36 and NPY3-36 which exert potent anorectic action by stimulating Y2 receptor (Y2R) function. This invites the possibility that DPP-IV inhibitors could be weight-neutral by preventing conversion of PYY/NPY to Y2R-selective peptide agonists. We therefore investigated whether co-administration of an Y2R-selective agonist could unmask potential weight lowering effects of the DDP-IV inhibitor linagliptin. Male diet-induced obese (DIO) mice received once daily subcutaneous treatment with linagliptin (3 mg/kg), a Y2R-selective PYY3-36 analogue (3 or 30 nmol/kg) or combination therapy for 14 days. While linagliptin promoted marginal weight loss without influencing food intake, the PYY3-36 analogue induced significant weight loss and transient suppression of food intake. Both compounds significantly improved oral glucose tolerance. Because combination treatment did not further improve weight loss and glucose tolerance in DIO mice, this suggests that potential negative modulatory effects of DPP-IV inhibitors on endogenous Y2R peptide agonist activity is likely insufficient to influence weight homeostasis. Weight-neutrality of DPP-IV inhibitors may therefore not be explained by counter-regulatory effects on PYY/NPY responses.

Effect of captopril on post-infarction remodelling visualized by light sheet microscopy and echocardiography.

Angiotensin converting enzyme inhibitors, among them captopril, improve survival following myocardial infarction (MI). The mechanisms of captopril action remain inadequately understood due to its diverse effects on multiple signalling pathways at different time periods following MI. Here we aimed to establish the role of captopril in late-stage post-MI remodelling. Left anterior descending artery (LAD) ligation or sham surgery was carried out in male C57BL/6J mice. Seven days post-surgery LAD ligated mice were allocated to daily vehicle or captopril treatment continued over four weeks. To provide comprehensive characterization of the changes in mouse heart following MI a 3D light sheet imaging method was established together with automated image analysis workflow. The combination of echocardiography and light sheet imaging enabled to assess cardiac function and the underlying morphological changes. We show that delayed captopril treatment does not affect infarct size but prevents left ventricle dilation and hypertrophy, resulting in improved ejection fraction. Quantification of lectin perfused blood vessels showed improved vascular density in the infarct border zone in captopril treated mice in comparison to vehicle dosed control mice. These results validate the applicability of combined echocardiographic and light sheet assessment of drug mode of action in preclinical cardiovascular research.

Whole-brain activation signatures of weight-lowering drugs.

OBJECTIVE: The development of effective anti-obesity therapeutics relies heavily on the ability to target specific brain homeostatic and hedonic mechanisms controlling body weight. To obtain further insight into neurocircuits recruited by anti-obesity drug treatment, the present study aimed to determine whole-brain activation signatures of six different weight-lowering drug classes. METHODS: Chow-fed C57BL/6J mice (n = 8 per group) received acute treatment with lorcaserin (7 mg/kg; i.p.), rimonabant (10 mg/kg; i.p.), bromocriptine (10 mg/kg; i.p.), sibutramine (10 mg/kg; p.o.), semaglutide (0.04 mg/kg; s.c.) or setmelanotide (4 mg/kg; s.c.). Brains were sampled two hours post-dosing and whole-brain neuronal activation patterns were analysed at single-cell resolution using c-Fos immunohistochemistry and automated quantitative three-dimensional (3D) imaging. RESULTS: The whole-brain analysis comprised 308 atlas-defined mouse brain areas. To enable fast and efficient data mining, a web-based 3D imaging data viewer was developed. All weight-lowering drugs demonstrated brain-wide responses with notable similarities in c-Fos expression signatures. Overlapping c-Fos responses were detected in discrete homeostatic and non-homeostatic feeding centres located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures as well as the dopaminergic system. CONCLUSIONS: Whole-brain c-Fos expression signatures of various weight-lowering drug classes point to a discrete set of brain regions and neurocircuits which could represent key neuroanatomical targets for future anti-obesity therapeutics.

Rodent models of diabetic kidney disease: human translatability and preclinical validity.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD). Except for SGLT2 inhibitors and GLP-1R agonists, there have been few changes in DKD treatment over the past 25 years, when multifactorial intervention was introduced in patients with type 2 diabetes mellitus (T2DM). The unmet clinical need is partly due to the lack of animal models that replicate clinical features of human DKD, which has raised concern about the utility of these models in preclinical drug discovery. In this review, we performed a comprehensive analysis of rodent models of DKD to compare treatment efficacy from preclinical testing with outcome from clinical trials. We also investigated whether rodent models are predictive for clinical outcomes of therapeutic agents in human DKD.

Transcriptomic changes in pancreatic islets, adipose and liver after Roux-en-Y gastric bypass in a diet-induced obese rat model.

Roux-en-Y gastric bypass (RYGB) is the most efficient intervention in morbid obesity and promotes metabolic improvements in several peripheral tissues. However, the underlying molecular mechanisms are still poorly understood. To further understand the effects of RYGB on peripheral tissues transcriptomes, we determined transcriptome signatures in pancreatic islets, adipose and liver tissue from diet-induced obese (DIO) rats model following RYGB. Whereas RYGB led to discrete gene expression changes in pancreatic islets, substantial transcriptome changes were observed in metabolic and immune signaling pathways in adipose tissue and the liver, indicating major gene adaptive responses in fat-storing tissues. Compared to RYGB DIO rats, peripheral tissue transcriptome signatures were markedly different in caloric restricted weight matching DIO rats, implying that caloric restriction paradigms do not reflect transcriptomic regulations of RYGB induced weight loss. The present gene expression study may serve as a basis for further investigations into molecular regulatory effects in peripheral tissues following RYGB-induced weight loss.

Identification and Metabolic Profiling of a Novel Human Gut-derived LEAP2 Fragment.

CONTEXT: The mechanisms underlying Roux-en-Y gastric bypass (RYGB) surgery-induced weight loss and the immediate postoperative beneficial metabolic effects associated with the operation remain uncertain. Enteroendocrine cell (EEC) secretory function has been proposed as a key factor in the marked metabolic benefits from RYGB surgery. OBJECTIVE: To identify novel gut-derived peptides with therapeutic potential in obesity and/or diabetes by profiling EEC-specific molecular changes in obese patients following RYGB-induced weight loss. SUBJECTS AND METHODS: Genome-wide expression analysis was performed in isolated human small intestinal EECs obtained from 20 gut-biopsied obese subjects before and after RYGB. Targets of interest were profiled for preclinical and clinical metabolic effects. RESULTS: Roux-en-Y gastric bypass consistently increased expression levels of the inverse ghrelin receptor agonist, liver-expressed antimicrobial peptide 2 (LEAP2). A secreted endogenous LEAP2 fragment (LEAP238-47) demonstrated robust insulinotropic properties, stimulating insulin release in human pancreatic islets comparable to the gut hormone glucagon-like peptide-1. LEAP238-47 showed reciprocal effects on growth hormone secretagogue receptor (GHSR) activity, suggesting that the insulinotropic action of the peptide may be directly linked to attenuation of tonic GHSR activity. The fragment was infused in healthy human individuals (n = 10), but no glucoregulatory effect was observed in the chosen dose as compared to placebo. CONCLUSIONS: Small intestinal LEAP2 expression was upregulated after RYGB. The corresponding circulating LEAP238-47 fragment demonstrated strong insulinotropic action in vitro but failed to elicit glucoregulatory effects in healthy human subjects.

Characterization of local gut microbiome and intestinal transcriptome responses to rosiglitazone treatment in diabetic db/db mice.

The gut microbiota has been implicated in the therapeutic effects of antidiabetics. It is unclear if antidiabetics directly influences gut microbiome-host interaction. Oral peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists, such as rosiglitazone, are potent insulin sensitizers used in the treatment of type 2 diabetes (T2D). PPAR-γ is abundantly expressed in the intestine, making it possible that PPAR-γ agonists directly influences gut microbiome-host homeostasis. The presented study therefore aimed to characterize local gut microbiome and intestinal transcriptome responses in diabetic db/db mice following rosiglitazone treatment. Diabetic B6.BKS(D)-Leprdb/J (db/db) mice (8 weeks of age) received oral dosing once daily with vehicle (n = 12) or rosiglitazone (3 mg/kg, n = 12) for 8 weeks. Gut segments (duodenum, jejunum, ileum, caecum, and colon) were sampled for paired analysis of gut microbiota and host transcriptome signatures using full-length bacterial 16S rRNA sequencing and RNA sequencing (n = 5-6 per group). Treatment with rosiglitazone improved glucose homeostasis without influencing local gut microbiome composition in db/db mice. In contrast, rosiglitazone promoted marked changes in ileal and colonic gene expression signatures associated with peroxisomal and mitochondrial lipid metabolism, carbohydrate utilization and immune regulation. In conclusion, rosiglitazone treatment markedly affected transcriptional markers of intestinal lipid metabolism and immune regulation but had no effect on the gut microbiome in diabetic db/db mice.

An Optimized Mouse Brain Atlas for Automated Mapping and Quantification of Neuronal Activity Using iDISCO+ and Light Sheet Fluorescence Microscopy.

In recent years, the combination of whole-brain immunolabelling, light sheet fluorescence microscopy (LSFM) and subsequent registration of data with a common reference atlas, has enabled 3D visualization and quantification of fluorescent markers or tracers in the adult mouse brain. Today, the common coordinate framework version 3 developed by the Allen's Institute of Brain Science (AIBS CCFv3), is widely used as the standard brain atlas for registration of LSFM data. However, the AIBS CCFv3 is based on histological processing and imaging modalities different from those used for LSFM imaging and consequently, the data differ in both tissue contrast and morphology. To improve the accuracy and speed by which LSFM-imaged whole-brain data can be registered and quantified, we have created an optimized digital mouse brain atlas based on immunolabelled and solvent-cleared brains. Compared to the AIBS CCFv3 atlas, our atlas resulted in faster and more accurate mapping of neuronal activity as measured by c-Fos expression, especially in the hindbrain. We further demonstrated utility of the LSFM atlas by comparing whole-brain quantitative changes in c-Fos expression following acute administration of semaglutide in lean and diet-induced obese mice. In combination with an improved algorithm for c-Fos detection, the LSFM atlas enables unbiased and computationally efficient characterization of drug effects on whole-brain neuronal activity patterns. In conclusion, we established an optimized reference atlas for more precise mapping of fluorescent markers, including c-Fos, in mouse brains processed for LSFM.

Gut Mucosal Gene Expression and Metabolic Changes After Roux-en-Y Gastric Bypass Surgery.

OBJECTIVE: Changes in the secretion of gut-derived peptide hormones have been associated with the metabolic benefits of Roux-en-Y gastric bypass (RYGB) surgery. In this study, the effects of RYGB on anthropometrics, postprandial plasma hormone responses, and mRNA expression in small intestinal mucosa biopsy specimens before and after RYGB were evaluated. METHODS: In a cross-sectional study, 20 individuals with obesity undergoing RYGB underwent mixed meal tests and upper enteroscopy with retrieval of small intestinal mucosa biopsy specimens 3 months before and after surgery. Concentrations of circulating gut and pancreatic hormones during mixed meal tests as well as full mRNA sequencing of biopsy specimens were evaluated. RESULTS: RYGB-induced improvements of body weight and composition, insulin resistance, and circulating cholesterols were accompanied by significant changes in postprandial plasma responses of pancreatic and gut hormones. Global gene expression analysis of biopsy specimens identified 2,437 differentially expressed genes after RYGB, including changes in genes that encode prohormones and G protein-coupled receptors. CONCLUSIONS: RYGB affects the transcription of a wide range of genes, indicating that the observed beneficial metabolic effects of RYGB may rely on a changed expression of several genes in the gut. RYGB-induced changes in the expression of genes encoding signaling peptides and G protein-coupled receptors may disclose new gut-derived treatment targets against obesity and diabetes.

3D quantification of changes in pancreatic islets in mouse models of diabetes type I and II.

Diabetes is characterized by rising levels of blood glucose and is often associated with a progressive loss of insulin-producing beta cells. Recent studies have demonstrated that it is possible to regenerate new beta cells through proliferation of existing beta cells or trans-differentiation of other cell types into beta cells, raising hope that diabetes can be cured through restoration of functional beta cell mass. Efficient quantification of beta cell mass and islet characteristics is needed to enhance drug discovery for diabetes. Here, we report a 3D quantitative imaging platform for unbiased evaluation of changes in islets in mouse models of type I and II diabetes. To determine whether the method can detect pharmacologically induced changes in beta cell volume, mice were treated for 14 days with either vehicle or the insulin receptor antagonist S961 (2.4 nmol/day) using osmotic minipumps. Mice treated with S961 displayed increased blood glucose and insulin levels. Light-sheet imaging of insulin and Ki67 (also known as Mki67)-immunostained pancreata revealed a 43% increase in beta cell volume and 21% increase in islet number. S961 treatment resulted in an increase in islets positive for the cell proliferation marker Ki67, suggesting that proliferation of existing beta cells underlies the expansion of total beta cell volume. Using light-sheet imaging of a non-obese diabetic mouse model of type I diabetes, we also characterized the infiltration of CD45 (also known as PTPRC)-labeled leukocytes in islets. At 14 weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. These results demonstrate how quantitative light-sheet imaging can capture changes in individual islets to help pharmacological research in diabetes.

Rat pancreatectomy combined with isoprenaline or uninephrectomy as models of diabetic cardiomyopathy or nephropathy.

Cardiovascular and renal complications are the predominant causes of morbidity and mortality amongst patients with diabetes. Development of novel treatments have been hampered by the lack of available animal models recapitulating the human disease. We hypothesized that experimental diabetes in rats combined with a cardiac or renal stressor, would mimic diabetic cardiomyopathy and nephropathy, respectively. Diabetes was surgically induced in male Sprague Dawley rats by 90% pancreatectomy (Px). Isoprenaline (Iso, 1 mg/kg, sc., 10 days) was administered 5 weeks after Px with the aim of inducing cardiomyopathy, and cardiac function and remodeling was assessed by echocardiography 10 weeks after surgery. Left ventricular (LV) fibrosis was quantified by Picro Sirius Red and gene expression analysis. Nephropathy was induced by Px combined with uninephrectomy (Px-UNx). Kidney function was assessed by measurement of glomerular filtration rate (GFR) and urine albumin excretion, and kidney injury was evaluated by histopathology and gene expression analysis. Px resulted in stable hyperglycemia, hypoinsulinemia, decreased C-peptide, and increased glycated hemoglobin (HbA1c) compared with sham-operated controls. Moreover, Px increased heart and LV weights and dimensions and caused a shift from α-myosin heavy chain (MHC) to ß-MHC gene expression. Isoprenaline treatment, but not Px, decreased ejection fraction and induced LV fibrosis. There was no apparent interaction between Px and Iso treatment. The superimposition of Px and UNx increased GFR, indicating hyperfiltration. Compared with sham-operated controls, Px-UNx induced albuminuria and increased urine markers of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and podocalyxin, concomitant with upregulated renal gene expression of NGAL and kidney injury molecule 1 (KIM-1). Whereas Px and isoprenaline separately produced clinical endpoints related to diabetic cardiomyopathy, the combination of the two did not accentuate disease development. Conversely, Px in combination with UNx resulted in several clinical hallmarks of diabetic nephropathy indicative of early disease development.

Human translatability of the GAN diet-induced obese mouse model of non-alcoholic steatohepatitis.

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.

Multi-omics characterization of a diet-induced obese model of non-alcoholic steatohepatitis.

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.