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This communication introduces the first-time application of high-resolution continuum-source molecular absorption spectrometry (HR CS MAS) for the quantification of a peptide. The graphite furnace technique was employed and the tripeptide glutathione (GSH) served as a model compound. Based on measuring sulfur in terms of carbon monosulfide (CS), a method was elaborated to analyze aqueous solutions of GSH. The most prominent wavelength of the CS molecule occurred at 258.0560 nm and was adduced for monitoring. The methodological development covered the optimization of the pyrolysis and vaporization temperatures. These were found optimally to be 250 °C and 2250 °C, respectively. Moreover, the effect of modifiers (zirconium, calcium, magnesium, palladium) on the absorption signals was investigated. The best results were obtained after permanent coating of the graphite tube with zirconium (total amount of 400 μg) and adding a combination of palladium (10 µL, 10 g L−1) and calcium (2 µL, 1 g L−1) as a chemical modifier to the probes (10 µL). Aqueous standard samples of GSH were used for the calibration. It showed a linear range of 2.5–100 µg mL−1 sulfur contained in GSH with a correlation coefficient R2 > 0.997. The developed method exhibited a limit of detection (LOD) and quantification (LOQ) of 2.1 µg mL−1 and 4.3 µg mL−1 sulfur, respectively. The characteristic mass accounted for 5.9 ng sulfur. The method confirmed the general suitability of MAS for the analysis of an oligopeptide. Thus, this study serves as groundwork for further development in order to extend the application of classical atomic absorption spectrometry (AAS).
Heart Rate Reduction by Ivabradine Improves Aortic Compliance in Apolipoprotein E-Deficient Mice
(2012)
Background: Impaired vascular compliance is associated with cardiovascular mortality. The effects of heart rate on vascular compliance are unclear. Therefore, we characterized effects of heart rate reduction (HRR) by I(f) current inhibition on aortic compliance and underlying molecular mechanisms in apolipoprotein E-deficient (ApoE<sup>–</sup>/<sup>–</sup>) mice. Methods: ApoE<sup>–</sup>/<sup>–</sup> mice fed a high-cholesterol diet and wild-type (WT) mice were treated with ivabradine (20 mg/kg/d) or vehicle for 6 weeks. Compliance of the ascending aorta was evaluated by MRI. Results: Ivabradine reduced heart rate by 113 ± 31 bpm (∼19%) in WT mice and by 133 ± 6 bpm (∼23%) in ApoE<sup>–</sup>/<sup>–</sup> mice. Compared to WT controls, ApoE<sup>–</sup>/<sup>–</sup> mice exhibited reduced distensibility and circumferential strain. HRR by ivabradine increased distensibility and circumferential strain in ApoE<sup>–</sup>/<sup>–</sup> mice but did not affect both parameters in WT mice. Ivabradine reduced aortic protein and mRNA expression of the angiotensin II type 1 (AT1) receptor and reduced rac1-GTPase activity in ApoE<sup>–</sup>/<sup>–</sup> mice. Moreover, membrane translocation of p47<sup>phox</sup> was inhibited. In ApoE<sup>–</sup>/<sup>–</sup> mice, HRR induced anti-inflammatory effects by reduction of aortic mRNA expression of IL-6, TNF-alpha and TGF-beta. Conclusion: HRR by ivabradine improves vascular compliance in ApoE<sup>–</sup>/<sup>–</sup> mice. Contributing mechanisms include downregulation of the AT1 receptor, attenuation of oxidative stress and modulation of inflammatory cytokine expression.
Gene Expression and Protein Abundance of Hepatic Drug Metabolizing Enzymes in Liver Pathology
(2021)
Transmembrane drug transport in hepatocytes is one of the major determinants of drug pharmacokinetics. In the present study, ABC transporters (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, and BSEP) and SLC transporters (MCT1, NTCP, OAT2, OATP1B1, OATP1B3, OATP2B1, OCT1, and OCT3) were quantified for protein abundance (LC-MS/MS) and mRNA levels (qRT-PCR) in hepatitis C virus (HCV)-infected liver samples from the Child–Pugh class A (n = 30), B (n = 21), and C (n = 7) patients. Protein levels of BSEP, MRP3, MCT1, OAT2, OATP1B3, and OCT3 were not significantly affected by HCV infection. P-gp, MRP1, BCRP, and OATP1B3 protein abundances were upregulated, whereas those of MRP2, MRP4, NTCP, OATP2B1, and OCT1 were downregulated in all HCV samples. The observed changes started to be seen in the Child–Pugh class A livers, i.e., upregulation of P-gp and MRP1 and downregulation of MRP2, MRP4, BCRP, and OATP1B3. In the case of NTCP, OATP2B1, and OCT1, a decrease in the protein levels was observed in the class B livers. In the class C livers, no other changes were noted than those in the class A and B patients. The results of the study demonstrate that drug transporter protein abundances are affected by the functional state of the liver in hepatitis C patients.
Membrane monocarboxylate transporter 1 (SLC16A1/MCT1) plays an important role in
hepatocyte homeostasis, as well as drug handling. However, there is no available information
about the impact of liver pathology on the transporter levels and function. The study was aimed to
quantify SLC16A1 mRNA (qRT-PCR) and MCT1 protein abundance (liquid chromatography–tandem
mass spectrometry (LC¬¬–MS/MS)) in the livers of patients diagnosed, according to the standard
clinical criteria, with hepatitis C, primary biliary cirrhosis, primary sclerosing hepatitis, alcoholic liver
disease (ALD), and autoimmune hepatitis. The stage of liver dysfunction was classified according to
Child–Pugh score. Downregulation of SLC16A1/MCT1 levels was observed in all liver pathology
states, significantly for ALD. The progression of liver dysfunction, from Child–Pugh class A to C,
involved the gradual decline in SLC16A1 mRNA and MCT1 protein abundance, reaching a clinically
significant decrease in class C livers. Reduced levels of MCT1 were associated with significant
intracellular lactate accumulation. The MCT1 transcript and protein did not demonstrate significant
correlations regardless of the liver pathology analyzed, as well as the disease stage, suggesting
posttranscriptional regulation, and several microRNAs were found as potential regulators of MCT1
abundance. MCT1 membrane immunolocalization without cytoplasmic retention was observed in all
studied liver pathologies. Overall, the study demonstrates that SLC16A1/MCT1 is involved in liver
pathology, especially in ALD
The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM).
In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans.
Dynamics of Vascular Protective and Immune Supportive Sphingosine-1-Phosphate During Cardiac Surgery
(2021)
Introduction
Sphingosine-1-phosphate (S1P) is a signaling lipid and crucial in vascular protection and immune response. S1P mediated processes involve regulation of the endothelial barrier, blood pressure and S1P is the only known inducer of lymphocyte migration. Low levels of circulatory S1P correlate with severe systemic inflammatory syndromes such as sepsis and shock states, which are associated with endothelial barrier breakdown and immunosuppression. We investigated whether S1P levels are affected by sterile inflammation induced by cardiac surgery.
Materials and Methods
In this prospective observational study we included 46 cardiac surgery patients, with cardiopulmonary bypass (CPB, n=31) and without CPB (off-pump, n=15). Serum-S1P, S1P-sources and carriers, von-Willebrand factor (vWF), C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured at baseline, post-surgery and at day 1 (POD 1) and day 4 (POD 4) after surgical stimulus.
Results
Median S1P levels at baseline were 0.77 nmol/mL (IQR 0.61-0.99) and dropped significantly post-surgery. S1P was lowest post-surgery with median levels of 0.37 nmol/mL (IQR 0.31-0.47) after CPB and 0.46 nmol/mL (IQR 0.36-0.51) after off-pump procedures (P<0.001). The decrease of S1P was independent of surgical technique and observed in all individuals. In patients, in which S1P levels did not recover to preoperative baseline ICU stay was longer and postoperative inflammation was more severe. S1P levels are associated with its sources and carriers and vWF, as a more specific endothelial injury marker, in different phases of the postoperative course. Determination of S1P levels during surgery suggested that also the anticoagulative effect of heparin might influence systemic S1P.
Discussion
In summary, serum-S1P levels are disrupted by major cardiac surgery. Low S1P levels post-surgery may play a role as a new marker for severity of cardiac surgery induced inflammation. Due to well-known protective effects of S1P, low S1P levels may further contribute to the observed prolonged ICU stay and worse clinical status. Moreover, we cannot exclude a potential inhibitory effect on circulating S1P levels by heparin anticoagulation during surgery, which would be a new pro-inflammatory pleiotropic effect of high dose heparin in patients undergoing cardiac surgery.
Neurosteroids, comprising pregnane, androstane, and sulfated steroids can alter neuronal excitability through interaction with ligand-gated ion channels and other receptors and have therefore a therapeutic potential in several brain disorders. They can be formed in brain cells or are synthesized by an endocrine gland and reach the brain by penetrating the blood–brain barrier (BBB). Especially sulfated steroids such as pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) depend on transporter proteins to cross membranes. In this review, we discuss the involvement of ATP-binding cassette (ABC)- and solute carrier (SLC)-type membrane proteins in the transport of these compounds at the BBB and in the choroid plexus (CP), but also in the secretion from neurons and glial cells. Among the ABC transporters, especially BCRP (ABCG2) and several MRP/ABCC subfamily members (MRP1, MRP4, MRP8) are expressed in the brain and known to efflux conjugated steroids. Furthermore, several SLC transporters have been shown to mediate cellular uptake of steroid sulfates. These include members of the OATP/SLCO subfamily, namely OATP1A2 and OATP2B1, as well as OAT3 (SLC22A3), which have been reported to be expressed at the BBB, in the CP and in part in neurons. Furthermore, a role of the organic solute transporter OSTα-OSTβ (SLC51A/B) in brain DHEAS/PregS homeostasis has been proposed. This transporter was reported to be localized especially in steroidogenic cells of the cerebellum and hippocampus. To date, the impact of transporters on neurosteroid homeostasis is still poorly understood. Further insights are desirable also with regard to the therapeutic potential of these compounds.
Background: Chronic kidney disease (CKD) and low serum total testosterone (TT) concentrations are independent predictors of mortality risk in the general population, but their combined potential for improved mortality risk stratification is unknown. Methods: We used data of 1,822 men from the population-based Study of Health in Pomerania followed- up for 9.9 years (median). The direct effects of kidney dysfunction (estimated glomerular filtration rate <60 ml/min/ 1.73 m<sup>2</sup>), albuminuria (urinary albumin-creatinine ratio ≧2.5 mg/mmol) and their combination (CKD) on all-cause and cardiovascular mortality were analyzed using multivariable Cox regression models. Serum TT concentrations below the age-specific 10th percentile (by decades) were considered low and were used for further risk stratification. Results: Kidney dysfunction (hazard ratio, HR, 1.40; 95% confidence interval, CI, 1.02–1.92), albuminuria (HR, 1.38; 95% CI, 1.06–1.79), and CKD (HR, 1.42; 95% CI, 1.09–1.84) were associated with increased all-cause mortality risk, while only kidney dysfunction (HR, 2.01; 95% CI, 1.21–3.34) was associated with increased cardiovascular mortality risk after multivariable adjustment. Men with kidney dysfunction and low TT concentrations were identified as high-risk individuals showing a more than 2-fold increased all-cause mortality risk (HR, 2.52; 95% CI, 1.08–5.85). Added to multivariable models, nonsignificant interaction terms suggest that kidney dysfunction and low TT are primarily additive rather than synergistic mortality risk factors. Conclusion: In the case of early loss of kidney function, measured TT concentrations might help to detect high-risk individuals for potential therapeutic interventions and to improve mortality risk assessment and outcome.
Abomasal emptying rate of diarrhoeic and healthy suckling calves fed with oral rehydration solutions
(2020)
Abstract
The aim of the study was to determine the abomasal emptying rate (AER) of calves suffering from naturally occurring diarrhoea compared with that of healthy calves. Furthermore, the effects of an oral rehydration solution (ORS) mixed into milk replacer on the AER were determined. Acetaminophen absorption test (APAT) was performed to estimate the AER. Sixty Holstein–Frisian calves (age < 14 days) were included in the study and divided into groups as follows: healthy calves (H; n = 16), healthy calves fed with ORS (HORS; n = 14), diarrhoeic calves (D; n = 15) and diarrhoeic calves fed with ORS (DORS; n = 15). For the APAT, the calves were fed 2 L of milk replacer containing 50 mg acetaminophen (AP)/kg body weight. Venous blood samples were collected before and after milk replacer and AP intake in 30–60 min intervals for 12 hr. During the APAT, no significant differences in median maximum acetaminophen concentration (Cmax) were observed among all groups. Time to reach maximum acetaminophen concentration (Tmax) in DORS (median 390 min, 25/75 quartiles: 300/480 min) was significantly higher compared with that in H (median: 270 min 25/75 quartiles: 210/315 min) and HORS (median: 300 min (25/75 quartiles: 240/360 min). Non‐linear regression revealed that the calculated abomasal half‐life (AP t1/2) tended to be delayed in DORS (median: 652 min, 25/75 quartiles: 445/795 min, p = .10). The area under the AP curve values (AUC) from 0 to 120 min and 0 to 240 min of the observation period were significantly higher in H than D and DORS. In conclusion, significant differences in the AER indices reflected delayed abomasal emptying in diarrhoeic calves. Furthermore, the hypertonic ORS tended to have an additive delaying impact on the AER, which needs attention for the feeding management of diarrhoeic calves.
Thiamine is substrate of the hepatic uptake transporter organic cation transporter 1 (OCT1), and pathological lipid metabolism was associated with OCT1‐dependent thiamine transport. However, it is unknown whether clinical pharmacokinetics of thiamine is modulated by OCT1 genotype. We analyzed thiamine transport in vitro, thiamine blood concentrations after high‐dose and low‐dose (nutritional) intake, and heritability of thiamine and thiamine‐phosphate blood concentrations. The variant OCT1*2 had reduced and OCT1*3 to OCT1*6 had deficient thiamine uptake activity. However, pharmacokinetics of thiamine did not differ depending on OCT1 genotype. Further studies in primary human hepatocytes indicated that several cation transporters, including OCT1, OCT3, and THTR‐2, contribute to hepatic uptake of thiamine. As much as 54% of the variation in thiamine and 75% in variation of thiamine monophosphate plasma concentrations was determined by heritable factors. Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research.
Genome-wide association studies have identified an association between isobutyrylcarnitine (IBC) and organic cation transporter 1 (OCT1) genotypes. Higher IBC blood concentrations in humans with active OCT1 genotypes and experimental studies with mouse OCT1 suggested an OCT1-mediated efflux of IBC. In this study, we wanted to confirm the suggested use of IBC as an endogenous biomarker of OCT1 activity and contribute to a better understanding of the mechanisms behind the association between blood concentrations of carnitine derivatives and OCT1 genotype. Blood and urine IBC concentrations were quantified in healthy volunteers regarding intra- and interindividual variation and correlation with OCT1 genotype and with pharmacokinetics of known OCT1 substrates. Furthermore, IBC formation and transport were studied in cell lines overexpressing OCT1 and its naturally occurring variants. Carriers of high-activity OCT1 genotypes had about 3-fold higher IBC blood concentrations and 2-fold higher amounts of IBC excreted in urine compared to deficient OCT1. This was likely due to OCT1 function, as indicated by the fact that IBC correlated with the pharmacokinetics of known OCT1 substrates, like fenoterol, and blood IBC concentrations declined with a 1 h time delay following peak concentrations of the OCT1 substrate sumatriptan. Thus, IBC is a suitable endogenous biomarker reflecting both, human OCT1 (hOCT1) genotype and activity. While murine OCT1 (mOCT1) was an efflux transporter of IBC, hOCT1 exhibited no IBC efflux activity. Inhibition experiments confirmed this data showing that IBC and other acylcarnitines, like butyrylcarnitine, 2-methylbutyrylcarnitine, and hexanoylcarnitine, showed reduced efflux upon inhibition of mOCT1 but not of hOCT1. IBC and other carnitine derivatives are endogenous biomarkers of hOCT1 genotype and phenotype. However, in contrast to mice, the mechanisms underlying the IBC-OCT1 correlation in humans is apparently not directly the OCT1-mediated efflux of IBC. A plausible explanation could be that hOCT1 mediates cellular concentrations of specific regulators or co-substrates in lipid and energy metabolism, which is supported by our in vitro finding that at baseline intracellular IBC concentration is about 6-fold lower alone by OCT1 overexpression.
In Germany, around 5.7 million people suffer from osteoporosis. Osteoporosis is characterised by a reduced bone mineral density that leads to an increased risk of fractures. The 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is an important regulator of local cortisol metabolism. It converts biologically inactive cortisone to biologically active cortisol, but can also catalyse the reverse reaction. 11β-HSD1 is strongly expressed in liver, but 11β-HSD1 expression and activity were also reported in bone. Moreover, polymorphisms in intron 5 of HSD11B1 (the gene encoding for 11β-HSD1) are associated with bone mineral density (BMD) and risk of fractures.
This work aimed to confirm and refine the associations between polymorphisms in intron 5 of HSD11B1 and BMD, and to identify the underlying molecular and cellular mechanisms. To this end, analyses were performed on three different levels:
i) studies in humans, to confirm and refine the association of polymorphisms in intron 5 of HSD11B1 with BMD, suppressed cortisol levels (PDC) and stiffness index,
ii) cellular analyses, to identify the role of 11β-HSD1 in differentiation of the immortalised human mesenchymal stem cell line SCP-1,
iii) molecular genetic analyses, to reveal the effect of intron 5 polymorphisms on transcriptional regulation.
Fine-mapping analyses of already existing clinical data from 452 osteoporosis patients (HSD study) did not point to another intron 5 SNP as being causative for the observed clinical association. A second prospective clinical study (OsteoGene) was performed to confirm the association of rs11811440 and rs932335 with PDC levels and BMD. A trend to decreased PDC levels and increased BMD was observed in homozygous carriers of the minor A-allele of rs11811440 in patients above the age of 65 years. Pooled analyses of the HSD and the OsteoGene studies revealed a significant association of the minor A-allele with increased Z-scores of the left femoral neck. No associations of rs11811440 and rs932335 with stiffness index, BMI and fat depots were detected the general population using data from the SHIP study.
To analyse the effect of 11β-HSD1 on differentiation of mesenchymal stem cells, HSD11B1 overexpressing and HSD11B1 knockout SCP-1 cells were generated. HSD11B1 was stably overexpressed in SCP-1 cells using targeted chromosomal integration. The successful overexpression was shown by 243-fold increased HSD11B1 mRNA expression levels and a 9 fold increased 11β-HSD1 activity, compared to the wildtype cells. Knockout cells were generated by CRISPR-Cas9 mediated gene editing targeting exon 2 and exon 5 of HSD11B1. Using next generation sequencing, the clones 1C4 and 2D10 were confirmed to carry two inactive HSD11B1 alleles and were chosen for further analyses. mRNA expression was unchanged in both knockout clones. However, a clear enzyme activity was detected in the 2D10 clone, whereas no cortisol production was detected in the 1C4 clone. SNaPshot analyses revealed the presence of wildtype cells in the 2D10 clone that became predominant with increased passages. Therefore, further analyses were focused on the 1C4 clone only. The protein expression in the 1C4 clone decreased to 30% of the expression of the wildtype cells.
HSD11B1 expression and cortisol production were compared between wildtype, knockout and overexpressing SCP-1 cells under three differentiation conditions: adipogenic, osteogenic with 1α,25-dihydroxyvitamin D3 and osteogenic with dexamethasone. HSD11B1 expression increased upon adipogenic differentiation and in the presence of cortisone in the wildtype and the overexpressing, but not in the knockout cells. Also, the cortisol production from cortisone increased over time in the overexpressing and the wildtype cells, but not in the knockout cells. The increase was dependent on the differentiation used between 3-fold and 9-fold higher in the overexpressing than in the wildtype cells.
The generated and validated overexpressing and knockout cell lines were used to analyse the influence of 11β-HSD1 on adipogenic and osteogenic differentiation. Upon adipogenic differentiation, the overexpressing cells accumulated significantly more lipid droplets than the wildtype cells. The accumulation of lipid droplets was not abolished in the knockout. However, when dexamethasone was substituted by cortisone, the knockout cells accumulated less lipid droplets than in the presence of dexamethasone, supporting the involvement of 11β-HSD1 in adipogenic differentiation. Expression of the adipogenic markers FABP4 and LPL increased upon adipogenic differentiation, but a distinct influence of the presence or absence of HSD11B1 on the FABP4 and LPL expression was not detected. Upon osteogenic differentiation with 1α,25-dihydroxyvitamin D3, ALP activity increased only in the knockout cells (more than 5-fold). Accordingly, the strongest increase in ALPL expression was detected also in the knockout cells. Both, ALP activity and gene expression were independent of cortisone. Addtionally, BGLAP expression was increased upon osteogenic differentiation. Unexpectedly, in the presence of cortisone, BGLAP expression increased in the overexpressing cells. Expression of the Wnt inhibitor DKK1 also increased in the overexpressing cells in the presence of cortisone indicating a decreased osteogenic differentiation. Moreover, expression of the adipogenic markers FABP4 and LPL increased in the overexpressing cells in the presence of cortisone indicating a switch from osteogenic to adipogenic differentiation. Upon osteogenic differentiation with dexamethasone, ALP activity and matrix mineralisation was lowest in the overexpressing cells.
Finally, the effects of the SNPs rs11811440, rs11119328, rs1000283 and rs932335 in intron 5 of HSD11B1 on transcriptional regulation were analysed by reporter gene assays and electrophoretic mobility shift assays. All four SNPs are genetically linked and are localized within evolutionary conserved regions. The minor C-allele of rs932335 significantly increased luciferase activity. In contrast, the major G-allele of rs932335 showed strong protein binding. However, no transcription factor binding sites were identified at the SNP sites. Additionally, bioinformatics analyses of publicly available RNA-Seq data of adipose tissue and liver confirmed the absence of alternative splicing. Alignment of HSD11B1 intron 5 to the Rfam database predicted the presence of non-coding RNAs (ncRNAs) in intron 5. However, none of the ncRNAs overlapped with the SNP sites.
In conclusion, 11β-HSD1 was shown to be involved in adipogenic differentiation and peripheral cortisol production by 11β-HSD1 promotes a switch from osteogenic to adipogenic differentiation. Moreover, among osteoporosis patients, homozygous carriers of the minor A-allele of rs11811440 have increased Z-scores of the femoral neck. Furthermore, HSD11B1 knockout and overexpressing cell lines were successfully generated and validated. These cell lines could be a useful tool in future analyses of the role of peripheral cortisol activation by 11β-HSD1 in differentiation of mesenchymal stem cells.
Osteoporosis, a complex chronic disease with increasing prevalence, is characterised by reduced bone mineral density (BMD) and increased fracture risk. The high heritability of BMD suggests substantial impact of the individual genetic disposition on bone phenotypes and the development of osteoporosis. In the past years, genome-wide association studies (GWAS) identified hundreds of genetic variants associated with BMD or osteoporosis. Here, we analysed 1103 single nucleotide polymorphisms (SNPs), previously identified as associated with estimated BMD (eBMD) in the UK Biobank. We assessed whether these SNPs are related to heel stiffness index obtained by quantitative ultrasound in 5665 adult participants of the Study of Health in Pomerania (SHIP). We confirmed 45 significant associations after correction for multiple testing. Next, we analysed six selected SNPs in 631 patients evaluated for osteoporosis [rs2707518 (CPED1/WNT16), rs3779381 (WNT16), rs115242848 (LOC101927709/EN1), rs10239787 (JAZF1), rs603424 (PKD2L1) and rs6968704 (JAZF1)]. Differences in minor allele frequencies (MAF) of rs2707518 and rs3779381 between SHIP participants (higher MAF) and patients evaluated for osteoporosis (lower MAF) indicated a protective effect of the minor allele on bone integrity. In contrast, differences in MAF of rs603424 indicated a harmful effect. Co-localisation analyses indicated that the rs603424 effect may be mediated via stearoyl-CoA desaturase (SCD) expression, an enzyme highly expressed in adipose tissue with a crucial role in lipogenesis. Taken together, our results support the role of the WNT16 pathway in the regulation of bone properties and indicate a novel causal role of SCD expression in adipose tissue on bone integrity.
Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells — a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.
Salivary glands provide secretory functions, including secretion of xenobiotics and among
them drugs. However, there is no published information about protein abundance of drug transporters
measured using reliable protein quantification methods. Therefore, mRNA expression and absolute
protein content of clinically relevant ABC (n = 6) and SLC (n = 15) family member transporters in the
human parotid gland, using the qRT-PCR and liquid chromatography-tandem mass spectrometry
(LC−MS/MS) method, were studied. The abundance of nearly all measured proteins ranged between
0.04 and 0.45 pmol/mg (OCT3 > MRP1 > PEPT2 > MRP4 > MATE1 > BCRP). mRNAs of ABCB1,
ABCC2, ABCC3, SLC10A1, SLC10A2, SLC22A1, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLCO1A2,
SLCO1B1, SLCO1B3 and SLCO2B1 were not detected. The present study provides, for the first time,
information about the protein abundance of membrane transporters in the human parotid gland,
which could further be used to define salivary bidirectional transport (absorption and secretion)
mechanisms of endogenous compounds and xenobiotics.
The multifunctional sphingosine-1-phosphate (S1P) is a lipid signaling molecule and central
regulator in the development of several cancer types. In recent years, intriguing information has
become available regarding the role of S1P in the progression of Glioblastoma multiforme (GBM),
the most aggressive and common brain tumor in adults. S1P modulates numerous cellular processes
in GBM, such as oncogenesis, proliferation and survival, invasion, migration, metastasis and stem cell
behavior. These processes are regulated via a family of five G-protein-coupled S1P receptors (S1PR1-5)
and may involve mainly unknown intracellular targets. Distinct expression patterns and multiple
intracellular signaling pathways of each S1PR subtype enable S1P to exert its pleiotropic cellular
actions. Several studies have demonstrated alterations in S1P levels, the involvement of S1PRs
and S1P metabolizing enzymes in GBM pathophysiology. While the tumorigenic actions of S1P
involve the activation of several kinases and transcription factors, the specific G-protein (Gi, Gq,
and G12/13)-coupled signaling pathways and downstream mediated effects in GBM remain to be
elucidated in detail. This review summarizes the recent findings concerning the role of S1P and its
receptors in GBM. We further highlight the current insights into the signaling pathways considered
fundamental for regulating the cellular processes in GMB and ultimately patient prognosis.