Control of (pre)-analytical aspects in immunoassay measurements of metabolic hormones in rodents

Bielohuby, Maximilian; Bidlingmaier, Martin; Schwahn, Uwe

doi:10.1530/ec-18-0035

Cited by 5 publications

(5 citation statements)

References 75 publications

(90 reference statements)

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“…All mice were sacrificed under isoflurane anesthesia by cervical dislocation after a 6 h fasting period. Blood samples were collected, processed and stored as previously recommended for metabolic rodent studies [16, 17]. In brief, blood for EDTA-plasma was collected from the retrobulbar sinus in chilled Eppendorf tubes which were pre-filled with EDTA and a protease inhibitor cocktail including a specific dpp4 inhibitor from Millipore (USA), a general protease-inhibitor from Sigma-Aldrich (USA) and an inhibitor of serine proteases (Roche Diagnostics; USA) to allow appropriate analysis of incretin hormones as previously described [16, 17].…”

Section: Methodsmentioning

confidence: 99%

A siRNA mediated hepatic dpp4 knockdown affects lipid, but not glucose metabolism in diabetic mice

Görgens

Jahn‐Hofmann

Bangari³

et al. 2019

PLoS ONE

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Systemic inhibition of dipeptidyl peptidase 4 (dpp4) represents an effective and established treatment option for type 2 diabetes (T2D). The current study investigated in mice if a liver selective knock-down of dpp4 by therapeutic siRNAs could be a novel, similarly effective treatment option for T2D. Furthermore, the potential effects on hepatic steatosis, inflammation and lipid metabolism were investigated after hepato-selective knock-down of dpp4. The knock-down efficiency and IC50 values of siRNAs targeting dpp4 were analyzed in PC3 cells. In two independent studies, either db/db mice or C57BL/6J mice were injected intravenously with a liposomal formulation of siRNAs targeting either dpp4 or a non-targeting control, followed by metabolically characterization. In comparator groups, additional cohorts of mice were treated with an oral dpp4 inhibitor. In both animal studies, we observed a robust knock-down (~75%) of hepatic dpp4 with a potent siRNA. Hepatic dpp4 knockdown did not significantly affect glucose metabolism or circulating incretin concentrations in both animal studies. However, in obese and diabetic db/db mice hepatic steatosis was reduced and hepatic mRNA expression of acaca, scd1, fasn and pparg was significantly lower after siRNA treatment. Systemic inhibition of the enzymatic dpp4 activity by an oral dpp4 inhibitor significantly improved glucose handling in db/db mice but did not affect hepatic endpoints. These data demonstrate that a targeted reduction of dpp4 expression in the liver may not be sufficient to improve whole-body glucose metabolism in obese and diabetic mice but may improve hepatic lipid metabolism.

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Section: Methodsmentioning

confidence: 99%

A siRNA mediated hepatic dpp4 knockdown affects lipid, but not glucose metabolism in diabetic mice

Görgens

Jahn‐Hofmann

Bangari³

et al. 2019

PLoS ONE

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“…It should be mentioned that further to the already discussed methodological limitations of this study (e.g., treatment duration, vehicle, potentially different culture conditions of cell lines), other factors that could not be controlled, such as features of the microbiome, epigenetic factors, or any additional unknowns as well as the inherent variability of hormone assays in the preanalytical and analytical phase (Bielohuby et al, 2018), could have contributed to the discrepant findings between our study and the study by Yore et al (2014).…”

Section: Limitations Of This Studymentioning

confidence: 99%

Acute and Repeated Treatment with 5-PAHSA or 9-PAHSA Isomers Does Not Improve Glucose Control in Mice

et al. 2018

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Fatty acid esters of hydroxylated fatty acids (FAHFAs) were discovered as a novel class of endogenous mammalian lipids whose profound effects on metabolism have been shown. In the current study, in vitro and in vivo the metabolic effects of two of these FAHFAs, namely palmitic acid-5- (or -9) -hydroxy-stearic acid (5- or 9-PAHSA, respectively) were profiled. In DIO mice fed with differentially composed low- or high-fat diets, acute and subchronic treatment with 5-PAHSA and 9-PAHSA alone, or in combination, did not significantly improve the deranged metabolic status. Neither racemic 5- or 9-PAHSA, nor the enantiomers were able to: (1) increase basal or insulin-stimulated glucose uptake in vitro, (2) stimulate GLP-1 release from GLUTag cells, or (3) induce GSIS in rat, mouse, or human islets or in a human pancreatic β cell line. Therefore, our data do not support the further development of PAHSAs or their derivatives for the control of insulin resistance and hyperglycemia.

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“…Homogenous variances were tested using the Levene test and heterogenous variances were tested using two‐way ANOVA. All blood samples were collected, processed and stored as previously recommended to minimize preanalytical variability of measurement parameters in metabolic rodent studies 25 …”

Section: Methodsmentioning

confidence: 99%

“…All blood samples were collected, processed and stored as previously recommended to minimize preanalytical variability of measurement parameters in metabolic rodent studies. 25 3 | RESULTS…”

Section: In Vivo Activity Profilementioning

confidence: 99%

Preclinical pharmacology of RA15127343: In vitro and in vivo activity of a novel ultralong‐acting basal insulin

Ulrich

Bielohuby

Korn

et al. 2022

Diabetes Obesity Metabolism

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Aim: To report the in vitro and in vivo preclinical pharmacokinetic (PK) and pharmacodynamic (PD) properties of RA15127343, a novel ultralong-acting insulin analogue targeting once-weekly administration, in female Göttingen minipigs. Methods:In vitro binding and activation of human insulin receptor isoforms (IR-A/IR-B), glucose uptake in rat myocytes, as well as mitogenic activity of RA15127343 were evaluated. In vivo, the PK and PD activities of RA15127343 were assessed in female, normoglycaemic Göttingen minipigs. The half-life (t 1/2 ) and time to maximum plasma concentration (T max ) of subcutaneously (SC) administered RA15127343 (10/30/45/60 nmol/kg) were estimated. In vivo blood glucose and endogenous plasma C-peptide concentrations after single SC administration (10/30/45/60 nmol/ kg) or repeated dosing (15 nmol/kg) were analysed. Results:In comparison to human insulin, RA15127343 showed lower in vitro binding affinity (19.9/6.31 μM vs. 1.10/1.14 nM) and activation (2.054 μM/669.6 nM vs. 26.04/18.24 nM) of IR-A/IR-B, lower potency to activate glucose uptake (855.2 vs. 3.37 nM) and lower mitogenic activity (17.92 μM vs. 10.78 nM; proliferation in MCF7 cells). In vivo, the mean t 1/2 and T max of RA15127343 after SC administration ranged from 48 to 59 and 30 to 39 hours, respectively. Blood glucose and plasma Cpeptide concentrations were significantly lower with RA15127343 (single/repeated doses) versus vehicle.Conclusions: RA15127343 showed an ultra-long t 1/2 with a slow onset of action.The preclinical pharmacological outcomes suggest RA15127343 could be a potential ultralong-acting insulin analogue with low risk of hypoglycaemia in humans.

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Control of (pre)-analytical aspects in immunoassay measurements of metabolic hormones in rodents

Cited by 5 publications

References 75 publications

A siRNA mediated hepatic dpp4 knockdown affects lipid, but not glucose metabolism in diabetic mice

A siRNA mediated hepatic dpp4 knockdown affects lipid, but not glucose metabolism in diabetic mice

Acute and Repeated Treatment with 5-PAHSA or 9-PAHSA Isomers Does Not Improve Glucose Control in Mice

Preclinical pharmacology of RA15127343: In vitro and in vivo activity of a novel ultralong‐acting basal insulin

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