| 1. |
- Kizilkaya, Hüsün S., et al.
(author)
-
Characterization of genetic variants of GIPR reveals a contribution of β-arrestin to metabolic phenotypes
- 2024
-
In: Nature Metabolism. - 2522-5812. ; 6:7, s. 1268-1281
-
Journal article (peer-reviewed)abstract
- Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes1. Yet both activation and inhibition of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in combination with glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) activation have resulted in similar clinical outcomes, as demonstrated by the GIPR–GLP-1R co-agonist tirzepatide2 and AMG-133 (ref. 3) combining GIPR antagonism with GLP-1R agonism. This underlines the importance of a better understanding of the GIP system. Here we show the necessity of β-arrestin recruitment for GIPR function, by combining in vitro pharmacological characterization of 47 GIPR variants with burden testing of clinical phenotypes and in vivo studies. Burden testing of variants with distinct ligand-binding capacity, Gs activation (cyclic adenosine monophosphate production) and β-arrestin 2 recruitment and internalization shows that unlike variants solely impaired in Gs signalling, variants impaired in both Gs and β-arrestin 2 recruitment contribute to lower adiposity-related traits. Endosomal Gs-mediated signalling of the variants shows a β-arrestin dependency and genetic ablation of β-arrestin 2 impairs cyclic adenosine monophosphate production and decreases GIP efficacy on glucose control in male mice. This study highlights a crucial impact of β-arrestins in regulating GIPR signalling and overall preservation of biological activity that may facilitate new developments in therapeutic targeting of the GIPR system.
|
|
| 2. |
- Di Martino, Elena, et al.
(author)
-
Inflammatory, metabolic, and sex-dependent gene-regulatory dynamics of microglia and macrophages in neonatal hippocampus after hypoxia-ischemia
- 2024
-
In: iScience. - : CELL PRESS. - 2589-0042. ; 27:4
-
Journal article (peer-reviewed)abstract
- Neonatal hypoxia-ischemia (HI) is a major cause of perinatal death and long-term disabilities worldwide. in the hippocampi of mice subjected to HI at postnatal day 9. Using inflammatory pathway and transcripcells isolated from hippocampi showed a partial convergence. Interestingly, microglia-specific genes in but not in females during the experimental time frame. These results highlight the importance of further investigations on metabolic rewiring to pave the way for future interventions in asphyxiated neonates.
|
|
| 3. |
- Koutsilieri, Stefania, et al.
(author)
-
Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models
- 2024
-
In: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 19:3
-
Journal article (peer-reviewed)abstract
- Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction-associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.
|
|
| 4. |
- Mickols, Evgeniya, et al.
(author)
-
OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids
- 2024
-
In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 14:1
-
Journal article (peer-reviewed)abstract
- 3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35–52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that can be performed using 3D PHH.
|
|
| 5. |
- Xing, Chen, et al.
(author)
-
The choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function
- 2024
-
In: Biotechnology Journal. - : John Wiley & Sons. - 1860-6768 .- 1860-7314. ; 19:2
-
Journal article (peer-reviewed)abstract
- Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments. We assessed the performance of ten different 384- and 96-well ultra-low attachment (ULA) microplates in facilitating three-dimensional spheroid culture of primary human hepatocytes (PHH) and primary canine hepatocytes (PCH) by examining effects on formation kinetics, spheroid morphology, long-term stability, functionality, expression signatures, and utility for hepatotoxicity assessment. Significant variations in experimental endpoints were observed between plates. These results can guide the optimization of spheroid experiments and contribute to the standardization of testing in three-dimensional organotypic liver cultures. image
|
|
