Ibuprofen, Flurbiprofen, Etoricoxib or Paracetamol Do Not Influence ACE2 Expression and Activity In Vitro or in Mice and Do Not Exacerbate In-Vitro SARS-CoV-2 Infection.

SARS-CoV-2 uses the human cell surface protein angiotensin converting enzyme 2 (ACE2) as the receptor by which it gains access into lung and other tissue. Early in the pandemic, there was speculation that a number of commonly used medications-including ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDs)-have the potential to upregulate ACE2, thereby possibly facilitating viral entry and increasing the severity of COVID-19. We investigated the influence of the NSAIDS with a range of cyclooxygenase (COX)1 and COX2 selectivity (ibuprofen, flurbiprofen, etoricoxib) and paracetamol on the level of ACE2 mRNA/protein expression and activity as well as their influence on SARS-CoV-2 infection levels in a Caco-2 cell model. We also analysed the ACE2 mRNA/protein levels and activity in lung, heart and aorta in ibuprofen treated mice. The drugs had no effect on ACE2 mRNA/protein expression and activity in the Caco-2 cell model. There was no up-regulation of ACE2 mRNA/protein expression and activity in lung, heart and aorta tissue in ibuprofen-treated mice in comparison to untreated mice. Viral load was significantly reduced by both flurbiprofen and ibuprofen at high concentrations. Ibuprofen, flurbiprofen, etoricoxib and paracetamol demonstrated no effects on ACE2 expression or activity in vitro or in vivo. Higher concentrations of ibuprofen and flurbiprofen reduced SARS-CoV-2 replication in vitro.

AMPKß1 and AMPKß2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, ß, and γ. AMPKα and ß are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKß1 and AMPKß2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKß2 was associated with a higher number of differentially regulated genes than the deletion of AMPKß1, suggesting that AMPKß2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKß2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKß1 impairs, whereas deficiency of AMPKß2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

The Novel Phosphate and Bile Acid Sequestrant Polymer SAR442357 Delays Disease Progression in a Rat Model of Diabetic Nephropathy.

As a gut-restricted, nonabsorbed therapy, polymeric bile acid sequestrants (BAS) play an important role in managing hyperlipidemia and hyperglycemia. Similarly, nonabsorbable sequestrants of dietary phosphate have been used for the management of hyperphosphatemia in end-stage renal disease. To evaluate the potential utility of such polymer sequestrants to treat type 2 diabetes (T2D) and its associated renal and cardiovascular complications, we synthesized a novel polymeric sequestrant, SAR442357, possessing optimized bile acid (BA) and phosphate sequestration characteristics. Long-term treatment of T2D obese cZucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) with SAR442357 resulted in enhanced sequestration of BAs and phosphate in the gut, improved glycemic control, lowering of serum cholesterol, and attenuation of diabetic kidney disease (DKD) progression. In comparison, colesevelam, a BAS with poor phosphate binding properties, did not prevent DKD progression, whereas losartan, an angiotensin II receptor blocker that is widely used to treat DKD, showed no effect on hyperglycemia. Analysis of hepatic gene expression levels of the animals treated with SAR442357 revealed upregulation of genes responsible for the biosynthesis of cholesterol and BAs, providing clear evidence of target engagement and mode of action of the new sequestrant. Additional hepatic gene expression pathway changes were indicative of an interruption of the enterohepatic BA cycle. Histopathological analysis of ZSF1 rat kidneys treated with SAR442357 further supported its nephroprotective properties. Collectively, these findings reveal the pharmacological benefit of simultaneous sequestration of BAs and phosphate in treating T2D and its associated comorbidities and cardiovascular complications. SIGNIFICANCE STATEMENT: A new nonabsorbed polymeric sequestrant with optimum phosphate and bile salt sequestration properties was developed as a treatment option for DKD. The new polymeric sequestrant offered combined pharmacological benefits including glucose regulation, lipid lowering, and attenuation of DKD progression in a single therapeutic agent.

Mastiha has efficacy in immune-mediated inflammatory diseases through a microRNA-155 Th17 dependent action.

Mastiha is a natural nutritional supplement with known anti-inflammatory properties. Non-alcoholic fatty liver disease (NAFLD) and Inflammatory bowel disease (IBD) are immune mediated inflammatory diseases that share common pathophysiological features. Mastiha has shown beneficial effects in both diseases. MicroRNAs have emerged as key regulators of inflammation and their modulation by phytochemicals have been extensively studied over the last years. Therefore, the aim of this study was to investigate whether a common route exists in the anti-inflammatory activity of Mastiha, specifically through the regulation of miRNA levels. Plasma miR-16, miR-21 and miR-155 were measured by Real-Time PCR before and after two double blinded and placebo-controlled randomized clinical trials with Mastiha. In IBD and particularly in ulcerative colitis patients in relapse, miR-155 increased in the placebo group (p = 0.054) whereas this increase was prevented by Mastiha. The mean changes were different in the two groups even after adjusting for age, sex and BMI (p = 0.024 for IBD and p = 0.042). Although the results were not so prominent in NAFLD, miR-155 displayed a downward trend in the placebo group (p = 0.054) whereas the levels did not changed significantly in the Mastiha group in patients with less advanced fibrosis. Our results propose a regulatory role for Mastiha in circulating levels of miR-155, a critical player in T helper-17 (Th17) differentiation and function.

Effects of Whole-Body Adenylyl Cyclase 5 (Adcy5) Deficiency on Systemic Insulin Sensitivity and Adipose Tissue.

Genome-wide association studies have identified adenylyl cyclase type 5 (ADCY5) as candidate gene for diabetes-related quantitative traits and an increased risk of type 2 diabetes. Mice with a whole-body deletion of Adcy5 (Adcy5-/-) do not develop obesity, glucose intolerance and insulin resistance, have improved cardiac function and increased longevity. Here, we investigated Adcy5 knockout mice (Adcy5-/-) to test the hypothesis that changes in adipose tissue (AT) may contribute to the reported healthier phenotype. In contrast to previous reports, we found that deletion of Adcy5 did not confer any physiological or biochemical benefits. However, this unexpected finding allowed us to investigate the effects of Adcy5 depletion on AT independently of lower body weight and a metabolically healthier phenotype. Adcy5-/- mice exhibited an increased number of smaller adipocytes, lower mean adipocyte size and a distinct AT gene expression pattern with midline 1 (Mid1) as the most significantly downregulated gene compared to control mice. Our Adcy5-/- model challenges previously described beneficial effects of Adcy5 deficiency and suggests that targeting Adcy5 does not improve insulin sensitivity and may therefore limit the relevance of ADCY5 as potential drug target.

Novel Inhibitors of Nicotinamide-N-Methyltransferase for the Treatment of Metabolic Disorders.

Nicotinamide-N-methyltransferase (NNMT) is a cytosolic enzyme catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to nicotinamide (Nam). It is expressed in many tissues including the liver, adipose tissue, and skeletal muscle. Its expression in several cancer cell lines has been widely discussed in the literature, and recent work established a link between NNMT expression and metabolic diseases. Here we describe our approach to identify potent small molecule inhibitors of NNMT featuring different binding modes as elucidated by X-ray crystallographic studies.

Incretin combination therapy for the treatment of non-alcoholic steatohepatitis.

AIMS: To test specific mono-agonists to the glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic peptide receptor (GIPR), individually and in combination, in a mouse model of diet-induced non-alcoholic steatohepatitis (NASH) and fibrosis in order to decipher the contribution of their activities and potential additive effects to improving systemic and hepatic metabolism. MATERIALS AND METHODS: We induced NASH by pre-feeding C57BL/6J mice a diet rich in fat, fructose and cholesterol for 36 weeks. This was followed by 8 weeks of treatment with the receptor-specific agonists 1-GCG (20 µg/kg twice daily), 2-GLP1 (3 µg/kg twice daily) or 3-GIP (30 µg/kg twice daily), or the dual (1 + 2) or triple (1 + 2 + 3) combinations thereof. A dual GLP-1R/GCGR agonistic peptide, 4-dual-GLP1/GCGR (30 µg/kg twice daily), and liraglutide (100 µg/kg twice daily) were included as references. RESULTS: Whereas low-dose 1-GCG or 3-GIP alone did not influence body weight, liver lipids and histology, their combination with 2-GLP1 provided additional weight loss, reduction in liver triglycerides and improvement in histological disease activity score. Notably, 4-dual-GLP-1R/GCGR and the triple combination of selective mono-agonists led to a significantly stronger reduction in the histological non-alcoholic fatty liver disease activity score compared to high-dose liraglutide, at the same extent of body weight loss. CONCLUSIONS: GCGR and GIPR agonism provide additional, body weight-independent improvements on top of GLP-1R agonism in a murine model of manifest NASH with fibrosis.

Identification and characterisation of carnostatine (SAN9812), a potent and selective carnosinase (CN1) inhibitor with in vivo activity.

Carnosinase 1 (CN1) has been postulated to be a susceptibility factor for developing diabetic nephropathy (DN). Although its major substrate, carnosine, is beneficial in rodent models of DN, translation of these findings to humans has been hampered by high CN1 activity in human serum resulting in rapid degradation of carnosine. To overcome this hurdle, we screened a protease-directed small-molecule library for inhibitors of human recombinant CN1. We identified SAN9812 as a potent and highly selective inhibitor of CN1 activity with a Ki of 11 nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30 mg/kg SAN9812 led to a sustained reduction in circulating CN1 activity in human CN1 transgenic (TG) mice. Simultaneous administration of carnosine and SAN9812 increased carnosine levels in plasma and kidney by up to 100-fold compared to treatment-naïve CN1-overexpressing mice. To our knowledge, this is the first study reporting on a potent and selective CN1 inhibitor with in vivo activity. SAN9812, also called carnostatine, may be used to increase renal carnosine concentration as a potential therapeutic modality for renal diseases linked to glycoxidative conditions.

Running on mixed fuel-dual agonistic approach of GLP-1 and GCG receptors leads to beneficial impact on body weight and blood glucose control: A comparative study between mice and non-human primates.

AIM: We performed acute and chronic studies in healthy and diet-induced obese animals using mouse-specific or monkey-specific dual GLP-1R/GCGR agonists to investigate their effects on food intake, body weight, blood glucose control and insulin secretion. The selective GLP-1R agonist liraglutide was used as comparator. METHODS: The mouse-specific dual agonist and liraglutide were tested in lean wild type, GLP-1R knockout and diet-induced obese mice at different doses. A chronic study was performed in DIO mice to investigate the effect on body weight, food consumption and total energy expenditure (TEE) in obese and diabetic monkeys with a focus on body weight and energy intake. RESULTS: The mouse-specific dual agonist and liraglutide similarly affected glycaemic control. A higher loss in body weight was measured in dual agonist-treated obese mice. The dual agonist significantly enhanced plasma glucose excursion in overnight fed GLP-1R-/- mice, probably reflecting a potent GCGR agonist activity. It increased TEE and enhanced fat and carbohydrate oxidation, while liraglutide produced no effect on TEE. In obese and diabetic monkeys, treatment with the monkey-specific dual agonist reduced total energy intake to 60%-70% of baseline TEI during chronic treatment. A decrease in body weight and significant improvement in glucose tolerance was observed. CONCLUSIONS: In DIO mice and non-human primates, dual agonists elicited robust glycaemic control, similar to the marketed GLP-1R agonist, while eliciting greater effects on body weight. Results from DIO mice suggest that the increase in TEE is caused not only by increased fat oxidation but also by an increase in carbohydrate oxidation.

Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase.

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ∼80% at 2 h when dosed in mice orally at 50 mg/kg.

GPR17 gene disruption does not alter food intake or glucose homeostasis in mice.

G protein-coupled receptor 17 (GPR17) was recently reported to be a Foxo1 target in agouti-related peptide (AGRP) neurons. Intracerebroventricular injection of GPR17 agonists induced food intake, whereas administration of an antagonist to the receptor reduced feeding. These data lead to the conclusion that pharmacological modulation of GPR17 has therapeutic potential to treat obesity. Here we report that mice deficient in Gpr17 (Gpr17(-/-)) have similar food intake and body weight compared with their wild-type littermates. Gpr17(-/-) mice have normal hypothalamic Agrp mRNA expression, AGRP plasma levels, and metabolic rate. GPR17 deficiency in mice did not affect glucose homeostasis or prevent fat-induced insulin resistance. These data do not support a role for GPR17 in the control of food intake, body weight, or glycemic control.

Crystal structures of monkey and mouse nicotinamide N-methyltransferase (NNMT) bound with end product, 1-methyl nicotinamide.

Nicotinamide N-methyltransferase (NNMT) is a S-adenosyl-l-methionine (SAM)-dependent enzyme that catalyzes N-methylation of nicotinamide (NA) and other pyridines to form N-methyl pyridinium ions. Here we report the first ternary complex X-ray crystal structures of monkey NNMT and mouse NNMT in bound form with the primary endogenous product, 1-methyl nicotinamide (MNA) and demethylated cofactor, S-adenosyl-homocysteine (SAH) determined at 2.30 Å and 1.88 Å respectively. The structural fold of these enzymes is identical to human NNMT. It is known that the primary endogenous product catalyzed by NNMT, MNA is a specific inhibitor of NNMT. Our data clearly indicates that the MNA binds to the active site and it would be trapped in the active site due to the formation of the bridge between the pole (long helix, α3) and long C-terminal loop. This might explain the mechanism of MNA acting as a feedback inhibitor of NNMT.

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling.

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 µM, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 µM RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.

Association of nicotinamide-N-methyltransferase mRNA expression in human adipose tissue and the plasma concentration of its product, 1-methylnicotinamide, with insulin resistance.

AIMS/HYPOTHESIS: Nicotinamide-N-methyltransferase (NNMT) was recently shown to be upregulated in mouse models of insulin resistance and obesity. So far, it is unknown whether NNMT is regulated in human disease. We have explored the hypothesis that white adipose tissue (WAT) NNMT expression and plasma 1-methylnicotinamide (MNA) concentration are increased in human insulin resistance and type 2 diabetes. METHODS: NNMT expression and plasma MNA were analysed in three groups of individuals: (1) 199 patients undergoing abdominal surgery; (2) 60 individuals on a 12-week exercise programme and (3) 55 patients on a two-step bariatric surgery programme. RESULTS: Patients with manifest type 2 diabetes have a significantly (approximately twofold) higher NNMT expression both in omental and subcutaneous WAT compared with controls. Notably, plasma MNA correlated significantly with WAT NNMT expression in patients with type 2 diabetes (women, r = 0.59, p < 0.001; men, r = 0.61, p < 0.001) but not in healthy control individuals. In insulin-resistant individuals, there was an inverse correlation between insulin sensitivity and plasma MNA (r = 0.44, p = 0.01) or adipose tissue NNMT mRNA (r = 0.64, p < 0.001). The latter association was confirmed in a second cohort (n = 60, r = 0.78, p < 0.001). Interventions improving insulin sensitivity--exercise and bariatric surgery--were associated with a significant (p < 0.001) reduction in WAT NNMT expression. Bariatric surgery was also associated with a significant decrease in plasma MNA. CONCLUSIONS/INTERPRETATION: We demonstrate that WAT NNMT expression is regulated in human insulin resistance and type 2 diabetes and that plasma MNA correlates with increased tissue NNMT expression and the degree of insulin resistance, making it a potential biomarker for loss of insulin sensitivity.

Serum carnosinase 1, an early indicator for incident microalbuminuria in type 1 diabetes.

Aims: Carnosinase (CN1) polymorphisms have been linked to diabetic kidney disease (DKD), as CN1 degrades dipeptides which scavenge oxidative metabolites and prevent the formation of advanced glycation end-products. In this work, we studied the association between serum CN1, the systemic redox status and long-term renal outcome in type 1 diabetes. Methods: Serum CN1 was measured in a prospective type 1 diabetes cohort (n = 218) with a 16-year follow-up. A total of 218 patients treated at the Diabetes Outpatient Clinic of the Weezenlanden Hospital (nowadays Isala Hospital, Zwolle, The Netherlands) were included in this analysis. We assessed whether serum CN1 was associated with renal function and development of DKD as well as other diabetic complications. Results: At baseline, age, systemic redox status and N-terminal pro brain-natriuretic peptide (NT-proBNP) were associated with serum CN1 concentration (p < 0.05). During follow-up, CN1 concentration in the middle tertile was associated with less incident microalbuminuria (odds ratio = 0.194, 95% C.I.: 0.049-0.772, p = 0.02) after adjustment for age, systemic redox status, NT-proBNP and sex. Discussion: Serum CN1 could predict incident microalbuminuria and may be used as a novel parameter to identify patients at risk for DKD.

An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Drug repurposing for the treatment of COVID-19: Targeting nafamostat to the lungs by a liposomal delivery system.

Despite tremendous global efforts since the beginning of the COVID-19 pandemic, still only a limited number of prophylactic and therapeutic options are available. Although vaccination is the most effective measure in preventing morbidity and mortality, there is a need for safe and effective post-infection treatment medication. In this study, we explored a pipeline of 21 potential candidates, examined in the Calu-3 cell line for their antiviral efficacy, for drug repurposing. Ralimetinib and nafamostat, clinically used drugs, have emerged as attractive candidates. Due to the inherent limitations of the selected drugs, we formulated targeted liposomes suitable for both systemic and intranasal administration. Non-targeted and targeted nafamostat liposomes (LipNaf) decorated with an Apolipoprotein B peptide (ApoB-P) as a specific lung-targeting ligand were successfully developed. The developed liposomal formulations of nafamostat were found to possess favorable physicochemical properties including nano size (119-147 nm), long-term stability of the normally rapidly degrading compound in aqueous solution, negligible leakage from the liposomes upon storage, and a neutral surface charge with low polydispersity index (PDI). Both nafamostat and ralimetinib liposomes showed good cellular uptake and lack of cytotoxicity, and non-targeted LipNaf demonstrated enhanced accumulation in the lungs following intranasal (IN) administration in non-infected mice. LipNaf retained its anti-SARS-CoV 2 activity in Calu 3 cells with only a modest decrease, exhibiting complete inhibition at concentrations >100 nM. IN, but not intraperitoneal (IP) treatment with targeted LipNaf resulted in a trend to reduced viral load in the lungs of K18-hACE2 mice compared to targeted empty Lip. Nevertheless, upon removal of outlier data, a statistically significant 1.9-fold reduction in viral load was achieved. This observation further highlights the importance of a targeted delivery into the respiratory tract. In summary, we were able to demonstrate a proof-of-concept of drug repurposing by liposomal formulations with anti-SARS-CoV-2 activity. The biodistribution and bioactivity studies with LipNaf suggest an IN or inhalation route of administration for optimal therapeutic efficacy.

Association Between Serum Carnosinase Concentration and Activity and Renal Function Impairment in a Type-2 Diabetes Cohort.

Introduction: Genetic studies have identified associations of carnosinase 1 (CN1) polymorphisms with diabetic kidney disease (DKD). However, CN1 levels and activities have not been assessed as diagnostic or prognostic markers of DKD in cohorts of patients with type 2 diabetes (T2D). Methods: We established high-throughput, automated CN1 activity and concentration assays using robotic systems. Using these methods, we determined baseline serum CN1 levels and activity in a T2D cohort with 970 patients with no or only mild renal impairment. The patients were followed for a mean of 1.2 years. Baseline serum CN1 concentration and activity were assessed as predictors of renal function impairment and incident albuminuria during follow up. Results: CN1 concentration was significantly associated with age, gender and estimated glomerular filtration rate (eGFR) at baseline. CN1 activity was significantly associated with glycated hemoglobin A1c (HbA1c) and eGFR. Serum CN1 at baseline was associated with eGFR decline and predicted renal function impairment and incident albuminuria during the follow-up. Discussion: Baseline serum CN1 levels were associated with presence and progression of renal function decline in a cohort of T2D patients. Confirmation in larger cohorts with longer follow-up observation periods will be required to fully establish CN1 as a biomarker of DKD.

Analysis of 16 studies in nine rodent models does not support the hypothesis that diabetic polyuria is a main reason of urinary bladder enlargement.

The urinary bladder is markedly enlarged in the type 1 diabetes mellitus model of streptozotocin-injected rats, which may contribute to the frequent diabetic uropathy. Much less data exists for models of type 2 diabetes. Diabetic polyuria has been proposed as the pathophysiological mechanism behind bladder enlargement. Therefore, we explored such a relationship across nine distinct rodent models of diabetes including seven models of type 2 diabetes/obesity by collecting data on bladder weight and blood glucose from 16 studies with 2-8 arms each; some studies included arms with various diets and/or pharmacological treatments. Data were analysed for bladder enlargement and for correlations between bladder weight on the one and glucose levels on the other hand. Our data confirm major bladder enlargement in streptozotocin rats and minor if any enlargement in fructose-fed rats, db/db mice and mice on a high-fat diet; enlargement was present in some of five not reported previously models. Bladder weight was correlated with blood glucose as a proxy for diabetic polyuria within some but not other models, but correlations were moderate to weak except for RIP-LCMV mice (r 2 of pooled data from all studies 0.0621). Insulin levels also failed to correlate to a meaningful extent. Various diets and medications (elafibranor, empagliflozin, linagliptin, semaglutide) had heterogeneous effects on bladder weight that often did not match their effects on glucose levels. We conclude that the presence and extent of bladder enlargement vary markedly across diabetes models, particularly type 2 diabetes models; our data do not support the idea that bladder enlargement is primarily driven by glucose levels/glucosuria.