Paraoxonase-2 agonist vutiglabridin promotes autophagy activation and mitochondrial function to alleviate non-alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Only limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH). Glabridin, a promising anti-obesity candidate, has only limited druggability due to its low in vivo chemical stability and bioavailability. Therefore, we developed vutiglabridin (VUTI), which is based on a glabridin backbone, and investigated its mechanism of action in treating NASH in animal models. EXPERIMENTAL APPROACH: Anti-NASH effects of VUTI were determined in in vitro fatty liver models, spheroids of primary human hepatocytes and L02 normal liver cell lines. To identify VUTI possible cellular target/s, biotin-labelled VUTI was synthesized and underwent chemical proteomic analysis. Further, the evaluation of VUTI therapeutic efficacy was carried out using an amylin-NASH and high-fat (HF) diet-induced obese (DIO) mouse models. This was carried out using transcriptomic, lipidomic and proteomic analyses of the livers from the amylin-NASH mouse model. KEY RESULTS: VUTI treatment markedly reduces hepatic steatosis, fibrosis and inflammation by promoting lipid catabolism, activating autophagy and improving mitochondrial dysfunction, all of which are hallmarks of effective NASH treatment. The cellular target of VUTI was identified as paraoxonase 2 (PON2), a newly proposed protein target for the treatment of NASH, VUTI enhanced PON2 activity. The results using PON2 knockdown cells demonstrated that PON2 is important for VUTI- activation of autophagy, promoting mitochondrial function, decreasing oxidative stress and alleviating lipid accumulation under lipotoxic condition. CONCLUSION AND IMPLICATIONS: Our data demonstrated that VUTI is a promising therapeutic for NASH. Targeting PON2 may be important for improving liver function in various immune-metabolic diseases including NASH.

Atlas of exercise-induced brain activation in mice.

OBJECTIVES: There is significant interest in uncovering the mechanisms through which exercise enhances cognition, memory, and mood, and lowers the risk of neurodegenerative diseases. In this study, we utilize forced treadmill running and distance-matched voluntary wheel running, coupled with light sheet 3D brain imaging and c-Fos immunohistochemistry, to generate a comprehensive atlas of exercise-induced brain activation in mice. METHODS: To investigate the effects of exercise on brain activity, we compared whole-brain activation profiles of mice subjected to treadmill running with mice subjected to distance-matched wheel running. Male mice were assigned to one of four groups: a) an acute bout of voluntary wheel running, b) confinement to a cage with a locked running wheel, c) forced treadmill running, or d) placement on an inactive treadmill. Immediately following each exercise or control intervention, blood samples were collected for plasma analysis, and brains were collected for whole-brain c-Fos quantification. RESULTS: Our dataset reveals 255 brain regions activated by acute exercise in mice, the majority of which have not previously been linked to exercise. We find a broad response of 140 regulated brain regions that are shared between voluntary wheel running and treadmill running, while 32 brain regions are uniquely regulated by wheel running and 83 brain regions uniquely regulated by treadmill running. In contrast to voluntary wheel running, forced treadmill running triggers activity in brain regions associated with stress, fear, and pain. CONCLUSIONS: Our findings demonstrate a significant overlap in neuronal activation signatures between voluntary wheel running and distance-matched forced treadmill running. However, our analysis also reveals notable differences and subtle nuances between these two widely used paradigms. The comprehensive dataset is accessible online at www.neuropedia.dk, with the aim of enabling future research directed towards unraveling the neurobiological response to exercise.

Improvement of NASH and liver fibrosis through modulation of the gut-liver axis by a novel intestinal FXR agonist.

Farnesoid X receptor (FXR) plays a pivotal role in the regulation of bile acid homeostasis and is involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Although FXR agonists effectively alleviate pathological features of NASH, adverse effects such as disturbance of cholesterol homeostasis and occurrence of pruritus remain to be addressed. Here, we identified a novel FXR agonist, ID119031166 (ID166), and explored the pharmacological benefits of ID166 in the treatment of NASH. ID166, a potent and selective non-bile acid FXR agonist, exhibits preferential distribution in the intestine and shows no agonist activity against potential itch receptors including Mas-related G protein-coupled receptor X4 (MRGPRX4). Interestingly, ID166 significantly attenuated total nonalcoholic fatty liver disease (NAFLD) activity and liver fibrosis in a free choice diet-induced NASH hamster model. In addition, ID166 drastically modulated the relative abundance of five gut microbes and reduced the increase in plasma total bile acid levels to normal levels in NASH hamsters. Moreover, long-term treatment with ID166 significantly improved key histological features of NASH and liver fibrosis in a diet-induced NASH mouse model. In the NASH mouse livers, RNA-seq analysis revealed that ID166 reduced the gene expression changes associated with both NASH and liver fibrosis. Notably, ID166 exhibited no substantial effects on scratching behavior and serum IL-31 levels in mice. Our findings suggest that ID166, a novel FXR agonist with improved pharmacological properties, provides a preclinical basis to optimize clinical benefits for NASH drug development.

Dietary intervention reverses molecular markers of hepatocellular senescence in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH.

BACKGROUND: Hepatocellular senescence may be a causal factor in the development and progression of non-alcoholic steatohepatitis (NASH). The most effective currently available treatment for NASH is lifestyle intervention, including dietary modification. This study aimed to evaluate the effects of dietary intervention on hallmarks of NASH and molecular signatures of hepatocellular senescence in the Gubra-Amylin NASH (GAN) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. METHODS: GAN DIO-NASH mice with liver biopsy-confirmed NASH and fibrosis received dietary intervention by switching to chow feeding (chow reversal) for 8, 16 or 24 weeks. Untreated GAN DIO-NASH mice and chow-fed C57BL/6J mice served as controls. Pre-to-post liver biopsy histology was performed for within-subject evaluation of NAFLD Activity Score and fibrosis stage. Terminal endpoints included blood/liver biochemistry, quantitative liver histology, mitochondrial respiration and RNA sequencing. RESULTS: Chow-reversal promoted substantial benefits on metabolic outcomes and liver histology, as demonstrated by robust weight loss, complete resolution of hepatomegaly, hypercholesterolemia, elevated transaminase levels and hepatic steatosis in addition to attenuation of inflammatory markers. Notably, all DIO-NASH mice demonstrated ≥ 2 point significant improvement in NAFLD Activity Score following dietary intervention. While not improving fibrosis stage, chow-reversal reduced quantitative fibrosis markers (PSR, collagen 1a1, α-SMA), concurrent with improved liver mitochondrial respiration, complete reversal of p21 overexpression, lowered γ-H2AX levels and widespread suppression of gene expression markers of hepatocellular senescence. CONCLUSIONS: Dietary intervention (chow reversal) substantially improves metabolic, biochemical and histological hallmarks of NASH and fibrosis in GAN DIO-NASH mice. These benefits were reflected by progressive clearance of senescent hepatocellular cells, making the model suitable for profiling potential senotherapeutics in preclinical drug discovery for NASH.

Semaglutide reduces tumor burden in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH-HCC with advanced fibrosis.

Non-alcoholic steatohepatitis (NASH) is emerging as a major cause of hepatocellular carcinoma (HCC), however, it is not resolved if compounds in late-stage clinical development for NASH may have additional therapeutic benefits in NASH-driven HCC (NASH-HCC). Here, we profiled monotherapy with semaglutide (glucagon-like-receptor-1 receptor agonist) and lanifibranor (pan-peroxisome proliferator-activated receptor agonist) in a diet-induced obese (DIO) mouse model of NASH-HCC. Disease progression was characterized in male C57BL/6 J mice fed the GAN (Gubra Amylin NASH) diet high in fat, fructose and cholesterol for 12-72 weeks (n = 15 per group). Other GAN DIO-NASH-HCC mice fed the GAN diet for 54 weeks and with biopsy-confirmed NASH (NAFLD Activity Score ≥ 5) and advanced fibrosis (stage F3) received vehicle (n = 16), semaglutide (30 nmol/kg, s.c., n = 15), or lanifibranor (30 mg/kg, p.o., n = 15) once daily for 14 weeks. GAN DIO-NASH-HCC mice demonstrated progressive NASH, fibrosis and HCC burden. Tumors presented with histological and molecular signatures of poor prognostic HCC. Consistent with clinical trial outcomes in NASH patients, both lanifibranor and semaglutide improved NASH while only lanifibranor reduced fibrosis in GAN DIO-NASH-HCC mice. Notably, only semaglutide reduced tumor burden in GAN DIO-NASH-HCC mice. In conclusion, the GAN DIO-NASH-HCC mouse is a clinical translational model of NASH-HCC. Semaglutide improves both NASH and tumor burden in GAN DIO-NASH-HCC mice, highlighting the suitability of this preclinical model for profiling novel drug therapies targeting NASH-HCC.

PPARγ lipodystrophy mutants reveal intermolecular interactions required for enhancer activation.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipocyte differentiation, and mutations that interfere with its function cause lipodystrophy. PPARγ is a highly modular protein, and structural studies indicate that PPARγ domains engage in several intra- and inter-molecular interactions. How these interactions modulate PPARγ's ability to activate target genes in a cellular context is currently poorly understood. Here we take advantage of two previously uncharacterized lipodystrophy mutations, R212Q and E379K, that are predicted to interfere with the interaction of the hinge of PPARγ with DNA and with the interaction of PPARγ ligand binding domain (LBD) with the DNA-binding domain (DBD) of the retinoid X receptor, respectively. Using biochemical and genome-wide approaches we show that these mutations impair PPARγ function on an overlapping subset of target enhancers. The hinge region-DNA interaction appears mostly important for binding and remodelling of target enhancers in inaccessible chromatin, whereas the PPARγ-LBD:RXR-DBD interface stabilizes the PPARγ:RXR:DNA ternary complex. Our data demonstrate how in-depth analyses of lipodystrophy mutants can unravel molecular mechanisms of PPARγ function.

GIP Affects Hepatic Fat and Brown Adipose Tissue Thermogenesis but Not White Adipose Tissue Transcriptome in Type 1 Diabetes.

CONTEXT: Glucose-dependent insulinotropic polypeptide (GIP) has been proposed to exert insulin-independent effects on lipid and bone metabolism. OBJECTIVE: We investigated the effects of a 6-day subcutaneous GIP infusion on circulating lipids, white adipose tissue (WAT), brown adipose tissue (BAT), hepatic fat content, inflammatory markers, respiratory exchange ratio (RER), and bone homeostasis in patients with type 1 diabetes. METHODS: In a randomized, placebo-controlled, double-blind, crossover study, 20 men with type 1 diabetes underwent a 6-day continuous subcutaneous infusion with GIP (6 pmol/kg/min) and placebo (saline), with an interposed 7-day washout period. RESULTS: During GIP infusion, participants (26 ± 8 years [mean ± SD]; BMI 23.8 ± 1.8 kg/m2; glycated hemoglobin A1c 51 ± 10 mmol/mol [6.8 ± 3.1%]) experienced transiently increased circulating concentrations of nonesterified fatty acid (NEFA) (P = 0.0005), decreased RER (P = 0.009), indication of increased fatty acid ß-oxidation, and decreased levels of the bone resorption marker C-terminal telopeptide (P = 0.000072) compared with placebo. After 6 days of GIP infusion, hepatic fat content was increased by 12.6% (P = 0.007) and supraclavicular skin temperature, a surrogate indicator of BAT activity, was increased by 0.29 °C (P < 0.000001) compared with placebo infusion. WAT transcriptomic profile as well as circulating lipid species, proteome, markers of inflammation, and bone homeostasis were unaffected. CONCLUSION: Six days of subcutaneous GIP infusion in men with type 1 diabetes transiently decreased bone resorption and increased NEFA and ß-oxidation. Further, hepatic fat content, and supraclavicular skin temperature were increased without affecting WAT transcriptomics, the circulating proteome, lipids, or inflammatory markers.

Nephroprotective Effects of Semaglutide as Mono- and Combination Treatment with Lisinopril in a Mouse Model of Hypertension-Accelerated Diabetic Kidney Disease.

Background: Obesity, hyperglycemia and hypertension are critical risk factors for development of diabetic kidney disease (DKD). Emerging evidence suggests that glucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular and renal outcomes in type 2 diabetes patients. Here, we characterized the effect of the long-acting GLP-1R agonist semaglutide alone and in combination with an ACE inhibitor (lisinopril) in a model of hypertension-accelerated, advanced DKD facilitated by adeno-associated virus-mediated renin overexpression (ReninAAV) in uninephrectomized (UNx) female diabetic db/db mice. Methods: Female db/db mice received a single intravenous injection of ReninAAV 1 week prior to UNx. Six weeks post-nephrectomy, db/db UNx-ReninAAV mice were administered (q.d.) vehicle, semaglutide (30 nmol/kg, s.c.) or semaglutide (30 nmol/kg, s.c.) + lisinopril (30 mg/kg, p.o.) for 11 weeks. Endpoints included blood pressure, plasma/urine biochemistry, kidney histopathology and RNA sequencing. Results: Vehicle-dosed db/db UNx-ReninAAV mice developed hallmarks of DKD characterized by severe albuminuria and advanced glomerulosclerosis. Semaglutide robustly reduced hyperglycemia, hypertension and albuminuria concurrent with notable improvements in glomerulosclerosis severity, podocyte filtration slit density, urine/renal kidney injury molecule-1 (KIM-1) levels and gene expression markers of inflammation and fibrogenesis in db/db UNx-ReninAAV mice. Co-administration of lisinopril further ameliorated hypertension and glomerulosclerosis. Conclusions: Semaglutide improves disease hallmarks in the db/db UNx-ReninAAV mouse model of advanced DKD. Further benefits on renal outcomes were obtained by adjunctive antihypertensive standard of care. Collectively, our study supports the development of semaglutide for management of DKD.

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.

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.

Chromatin Immunoprecipitation for Identification of Protein-DNA Interactions in Human Cells.

Chromatin immunoprecipitation (ChIP) is a powerful technique allowing for investigation of protein-DNA interactions in living cells. Here, we provide a detailed step-by-step protocol for ChIP and highlight important considerations, challenges and pitfalls often encountered in the ChIP procedure. Furthermore, we present data of key quality control (QC) steps and exemplify material performance validation on transcription factor ChIP to provide a QC guide for setting up ChIP. Finally, we provide guidelines for scaling of the ChIP procedure to ChIP sequencing (ChIP-seq) and discuss important considerations associated with this.