18 research outputs found
Establishing a Reliable Multiple Reaction Monitoring-Based Method for the Quantification of Obesity-Associated Comorbidities in Serum and Adipose Tissue Requires Intensive Clinical Validation
Multiple reaction monitoring (MRM)-based mass spectrometric quantification
of peptides and their corresponding proteins has been successfully
applied for biomarker validation in serum. The option of multiplexing
offers the chance to analyze various proteins in parallel, which is
especially important in obesity research. Here, biomarkers that reflect
multiple comorbidities and allow monitoring of therapy outcomes are
required. Besides the suitability of established MRM assays for serum
protein quantification, it is also feasible for analysis of tissues
secreting the markers of interest. Surprisingly, studies comparing
MRM data sets with established methods are rare, and therefore the
biological and clinical value of most analytes remains questionable.
A MRM method using nano-UPLC-MS/MS for the quantification of obesity
related surrogate markers for several comorbidities in serum, plasma,
visceral and subcutaneous adipose tissue was established. Proteotypic
peptides for complement C3, adiponectin, angiotensinogen, and plasma
retinol binding protein (RBP4) were quantified using isotopic dilution
analysis and compared to the standard ELISA method. MRM method variabilities
were mainly below 10%. The comparison with other MS-based approaches
showed a good correlation. However, large differences in absolute
quantification for complement C3 and adiponectin were obtained compared
to ELISA, while less marked differences were observed for angiotensinogen
and RBP4. The verification of MRM in obesity was performed to discriminate
first lean and obese phenotype and second to monitor excessive weight
loss after gastric bypass surgery in a seven-month follow-up. The
presented MRM assay was able to discriminate obese phenotype from
lean and monitor weight loss related changes of surrogate markers.
However, inclusion of additional biomarkers was necessary to interpret
the MRM data on obesity phenotype properly. In summary, the development
of disease-related MRMs should include a step of matching the MRM
data with clinically approved standard methods and defining reference
values in well-sized representative age, gender, and disease-matched
cohorts
Establishing a Reliable Multiple Reaction Monitoring-Based Method for the Quantification of Obesity-Associated Comorbidities in Serum and Adipose Tissue Requires Intensive Clinical Validation
Multiple reaction monitoring (MRM)-based mass spectrometric quantification
of peptides and their corresponding proteins has been successfully
applied for biomarker validation in serum. The option of multiplexing
offers the chance to analyze various proteins in parallel, which is
especially important in obesity research. Here, biomarkers that reflect
multiple comorbidities and allow monitoring of therapy outcomes are
required. Besides the suitability of established MRM assays for serum
protein quantification, it is also feasible for analysis of tissues
secreting the markers of interest. Surprisingly, studies comparing
MRM data sets with established methods are rare, and therefore the
biological and clinical value of most analytes remains questionable.
A MRM method using nano-UPLC-MS/MS for the quantification of obesity
related surrogate markers for several comorbidities in serum, plasma,
visceral and subcutaneous adipose tissue was established. Proteotypic
peptides for complement C3, adiponectin, angiotensinogen, and plasma
retinol binding protein (RBP4) were quantified using isotopic dilution
analysis and compared to the standard ELISA method. MRM method variabilities
were mainly below 10%. The comparison with other MS-based approaches
showed a good correlation. However, large differences in absolute
quantification for complement C3 and adiponectin were obtained compared
to ELISA, while less marked differences were observed for angiotensinogen
and RBP4. The verification of MRM in obesity was performed to discriminate
first lean and obese phenotype and second to monitor excessive weight
loss after gastric bypass surgery in a seven-month follow-up. The
presented MRM assay was able to discriminate obese phenotype from
lean and monitor weight loss related changes of surrogate markers.
However, inclusion of additional biomarkers was necessary to interpret
the MRM data on obesity phenotype properly. In summary, the development
of disease-related MRMs should include a step of matching the MRM
data with clinically approved standard methods and defining reference
values in well-sized representative age, gender, and disease-matched
cohorts
Univariate correlations between <i>ADCY5</i> mRNA expression in visceral (VAT) and subcutaneous (SC) adipose tissue and parameters of obesity, insulin sensitivity, and inflammation.
<p>R<sup>2</sup>, PearsonâČs correlation coefficient. Analyses were performed only in individuals for which complete data sets were available (n = 240). Significant associations are highlighted in bold if they remained significant after adjusting for age, gender and BMI (in brackets: p-values adjusted for BMI, age, and gender). Significant associations after adjustment are highlighted in bold. (data are log transformed). FPG, fasting plasma glucose; n.a., not applicable.</p><p>Univariate correlations between <i>ADCY5</i> mRNA expression in visceral (VAT) and subcutaneous (SC) adipose tissue and parameters of obesity, insulin sensitivity, and inflammation.</p
<i>Adcy5</i> mRNA expression in adipose tissue of C57BL/6 mice on either chow or high fat diet (HFD).
<p><i>Adcy5</i> mRNA expression was calculated relative to the expression of <i>18S rRNA</i> (AU = arbitrary units). *p<0.05; **p<0.01 for comparisons between chow and HFD; #p<0.05 for comparisons between fat depots. SAT, subcutaneous adipose tissue; EPI AT, epigonadal adipose tissue.</p
Anthropometric and metabolic characteristics of participants in the study of adipose tissue <i>ADCY5</i> mRNA expression (n = 244).
<p>Data are means ± SD.</p><p>*p<0.05,</p><p>**p<0.01,</p><p>***p<0.001 for comparisons between men and women, individuals with T2D versus no diabetes or lean versus obese.</p><p>BMI, body mass index, WHR, waist-hip ratio, 2h oGTT, 2 hour oral glucose tolerance test, GIR, glucose infusion rate, hsCRP, high sensitive C-reactive protein, HDL, high-density lipoprotein, LDL, how-density lipoprotein, SC subcutaneous</p><p>Anthropometric and metabolic characteristics of participants in the study of adipose tissue <i>ADCY5</i> mRNA expression (n = 244).</p
Anthropometric and metabolic characteristics of participants in the study of adipose tissue <i>ADCY5</i> mRNA expression according to the rs11708067 genotype.
<p>Data are means ± SD.</p><p>*p<0.05 for comparisons between AA and AG genotypes,</p><p><sup>#</sup>p<0.05 for comparisons between AA and GG genotypes,</p><p><sup>§</sup> p<0.05 for comparisons between AG and GG genotypes.</p><p>BMI, body mass index, WHR, waist-hip ratio, 2h oGTT, 2 hour oral glucose tolerance test, GIR, glucose infusion rate, hsCRP, high sensitive C-reactive protein, HDL, high-density lipoprotein, LDL, low-density lipoprotein, SC subcutaneous, SAT, subcutaneous adipose tissue, VAT, visceral adipose tissue</p><p>Anthropometric and metabolic characteristics of participants in the study of adipose tissue <i>ADCY5</i> mRNA expression according to the rs11708067 genotype.</p
Hepatocyte-specific knockdown of PPP2R5C leads to increased lipogenesis and lipid secretion.
<p><b>(A-C)</b> Liver-specific knockdown of PPP2R5C leads to pro-anabolic changes including increased liver lipid synthesis and secretion and reduced glycogen breakdown. As in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.g002" target="_blank">Fig 2</a>, 7 weeks post hepatocyte-specific knockdown, liver weight (A), glycogen (B) and triglycerides (C) were quantified. (n = 5 or 6) <b>(D-E)</b> Cellular triglyceride levels are increased in Hepa 1â6 (D) or mouse primary hepatocytes (E) upon PPP2R5C knockdown. Cells infected as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.g002" target="_blank">Fig 2</a>. (n = 3) (<b>F-G</b>) Liver-specific knockdown of PPP2R5C leads to elevated serum VLDL levels. 7 weeks post hepatocyte-specific knockdown, serum triglycerides were quantified either in aggregate (F), or when fractionated by FPLC to resolve lipoprotein particles of various densities (G). (n = 5 or 6) Error bars: std. dev. *p-value<0.05, **p-value<0.01 by one-way ANCOVA with liver NEFA as a covariate (C, âRandomâ) or student t-test (A-F).</p
Evaluation of Transient Elastography, Acoustic Radiation Force Impulse Imaging (ARFI), and Enhanced Liver Function (ELF) Score for Detection of Fibrosis in Morbidly Obese Patients
<div><p>Background</p><p>Liver fibrosis induced by non-alcoholic fatty liver disease causes peri-interventional complications in morbidly obese patients. We determined the performance of transient elastography (TE), acoustic radiation force impulse (ARFI) imaging, and enhanced liver fibrosis (ELF) score for fibrosis detection in bariatric patients.</p><p>Patients and Methods</p><p>41 patients (median BMI 47 kg/m<sup>2</sup>) underwent 14-day low-energy diets to improve conditions prior to bariatric surgery (day 0). TE (M and XL probe), ARFI, and ELF score were performed on days -15 and -1 and compared with intraoperative liver biopsies (NAS staging).</p><p>Results</p><p>Valid TE and ARFI results at day -15 and -1 were obtained in 49%/88% and 51%/90% of cases, respectively. High skin-to-liver-capsule distances correlated with invalid TE measurements. Fibrosis of liver biopsies was staged as F1 and F3 in n = 40 and n = 1 individuals. However, variations (median/range at d-15/-1) of TE (4.6/2.6â75 and 6.7/2.9â21.3 kPa) and ARFI (2.1/0.7â3.7 and 2.0/0.7â3.8 m/s) were high and associated with overestimation of fibrosis. The ELF score correctly classified 87.5% of patients.</p><p>Conclusion</p><p>In bariatric patients, performance of TE and ARFI was poor and did not improve after weight loss. The ELF score correctly classified the majority of cases and should be further evaluated.</p></div
PPP2R5C HepKD in <i>db/db</i> mouse liver improves insulin sensitivity, decreases hyperglycemia, but worsens the dyslipidemia.
<p><b>(A)</b> Hyperglycemia in <i>db/db</i> mice is decreased upon hypatocyte-specific PPP2R5C knockdown with adeno-associated virus. After 5 weeks knockdown, <i>db/db</i> mice were sacrificed under <i>ad libitum</i> feeding with normal chow diet. Blood glucose was monitored at each week, week 0 was one day before virus injection (n = 6). <b>(B)</b> Insulin tolerance test shows improved insulin sensitivity in PPP2R5C knockdown <i>db/db</i> mice at week 4 after virus injection (1.5IU/kg insulin was tail-injected after 6 hour fasting) (n = 6). <b>(C-F)</b> PPP2R5C HepKD in <i>db/db</i> mice increases body weight (C), whole body fat content (D), and liver weight (E), without changing abdominal adipose tissue weight (Abd.WAT) (F) (n = 6). Error bars: std. dev. *p-value<0.05 and **p-value<0.01 by student t-test (D-E). p-value in the (A-C) was calculated by two-way ANOVA.</p
Hepatocyte-specific knockdown of PPP2R5C leads to increased glucose uptake and improved glucose tolerance.
<p><b>(A-C)</b> Increased glucose uptake/tolerance in mice upon liver-specific knockdown of PPP2R5C. 8â10 week CL57BL/6 male mice tail-injected with adeno-associated virus containing miRNA targeting PPP2R5C (âPPP2R5C KDâ) or a scrambled control miRNA (âControl KDâ). 7 weeks after knockdown mice were starved for 16 hours (âFastingâ) or starved and then given normal chow diet for 6 hours (âRefedâ) prior to sacrificing. Although blood glucose levels are not altered (A), serum insulin levels are significantly reduced (B) compared to controls. (C) Glucose tolerance test performed 4 weeks after virus injection shows significantly improved tolerance in knockdown mice (2g glucose/kg body weight injected intraperitoneally, n = 12). <b>(D)</b> Insulin signaling, detected via AKT and GSK3 beta phosphorylation, does not drop in all feeding regimes in PPP2R5C HepKD livers compared to controls, despite PPP2R5C HepKD serum insulin levels being lower (see panel B). <b>(E)</b> Insulin sensitivity is increased after PPP2R5C knockdown in liver. Control C57BL/6 mice and PPP2R5C HepKD mice were virus-injected as in Fig 2. Four weeks later, mice were fasted for 6 hours, then insulin was injected 1IU/kg and 10 minutes later mice were sacrified and liver samples were taken. <b>(F-G)</b> Glucose consumption and lactate production are increased in Hepa 1â6 cells upon PPP2R5C knockdown. Hepa 1â6 cells infected by adenovirus carrying shRNA targeting all mouse PPP2R5C isoforms (PPP2R5C KD) or a negative-control scramble shRNA (Control KD). After 48h, glucose consumption (F) and lactate production (G) were measured in the medium for 24 hours, and normalized to total cell protein. (n = 3) <b>(H)</b> Glycolytic flux measured as ECAR (extracellular acidification rate) using the glycolysis stress kit from Seahorse Bioscience on the extracellular flux analyser XF96. After addition of glucose to control or PPP2R5C knockdown Hepa 1â6 cells, oligomycin is added to inhibit respiration, thereby boosting glycolytic flux. 2-deoxy-glucose is added to compete with glucose and shut down glycolysis (n = 9). <b>(I)</b> Acute glucose uptake is increased in Hepa 1â6 cells upon PPP2R5C knockdown. Stably transfected Hepa1-6 cell-lines carrying two independent, inducible shRNAs (PPP2R5C KD1 and KD2) were induced with 30 ÎŒg/ml cumate for 3 days, starved overnight in serum-free DMEM, and uptake of fluorescent 2-deoxy-glucose analog 2-NBDG was quantified by FACS. (n = 3) Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001, â p-value<10<sup>â4</sup> by Wilcoxon signed-rank test (C) or student t-test (B, F-I).</p