Quantitative (phospho)-proteomics identifies a novel role for hedgehog in reprogramming adipocyte energy metabolism
Bibliographic record
Abstract
Hedgehog (HH) signaling plays a fundamental role in developmental processes conserved from flies to humans, whereas aberrant HH reactivation has been linked to multiple cancer types.Recent data from our laboratory identified (i) a key role for HH in inhibiting white but not brown adipocyte differentiation in vitro and in vivo, and (ii) that this inhibitory HH effect on white fat cell differentiation can be blocked by the activated T-cell cytokine interferon-gamma (IFN-γ).However, no studies have so far addressed the impact of HH on mature adipocytes and no data at all are available on HH induced (phospho)-proteomic changes.Therefore, we set out to address both aspects and made use of quantitative proteomic approaches to identify for the first time new HH targets and mechanisms in mature adipocytes.We performed 2D-PAGE and quantitative label-free 1D-GeLC/MSMS with (i) phosphoprotein enriched samples; and (ii) cytoplasmic fractions to gain unbiased insight into rapid phosphorylation and lasting expression changes, respectively.Western blots were performed to validate results.Cellular metabolites were measured by ELISA.Phospho-protein-specific 1D-and 2D-gels revealed a highly reproducible phosphorylation of pyruvate dehydrogenase (PDH) E1 alpha in HH treated samples.In line with this data we observed a significant up-regulation of PDH-kinase in the cytoplasmic fraction.Furthermore, the glycolytic key enzymes phosphofructokinase (PFK) and pyruvate kinase M2 (PKM2) were significantly altered in HH treated samples, leading to a shift towards lactate production.Pathway analyses identified major changes in glycolysis and mitochondrial function (OXPHOS, TCA cycle).Of note, increased NAD + /NADH and NADP + /NADPH ratios further reflected HH induced metabolic reprogramming.In summary, our unbiased (phospho)-proteomic screen identified HH as a hitherto unknown regulator of cellular energy metabolism, leading to increased glucose turnover and lactate production.IV ZUSAMMENFASSUNG Der sogenannte Hedgehog Signaltransduktionsweg (HH-STW) spielt eine essentielle Rolle in Entwicklungsprozessen von der Fruchtfliege bis hin zum Mensch.Eine unzureichende Funktion des HH-STW führt zu schweren Missbildungen, wobei eine Überreaktion mit Tumorentstehung in Verbindung steht.Rezente Daten unserer Forschungsarbeiten bestätigten (i) eine Schlüsselfunktion des HH-STW in der Inhibierung der Differenzierung weißer, aber nicht brauner Fettzellen sowohl in vitro als auch in vivo und (ii) dass diese hemmende Wirkung durch das aktivierte T-Zellen Zytokin Interferon-gamma (IFN-γ) blockiert wird.Interessanterweise hat sich bis dato keine Studie damit befasst, den HH-STW in reifen Fettzellen zu untersuchen.Auch sind keine relevanten (phospho)-proteomischen Daten vorhanden.Somit setzten wir uns das Ziel, als erste Forschungsgruppe weltweit, neue Mechanismen des HH-STW auf reife Fettzellen, mit Hilfe quantitativer proteomischer Methoden zu erfassen.2D-PAGE und quantitative label-free 1D-GeLC/MSMS mit (i) angereicherten Phosphoproteinen und (ii) der zytoplasmatischen Fraktion reifer Adipozyten wurde angewandt.Ziel war es, Änderungen in (De)-Phosphorylierungen beziehungsweise des Expressionsmusters in Hedgehog (HH) stimulierten Fettzellen zu detektieren.Western blot Analysen, sowie ELISA Messungen zellulärer Metaboliten untermauerten diese Ergebnisse.Phosphoprotein-spezifische 1D-und 2D-Gele identifizierten eine hoch reproduzierbare Phosphorylierung der Pyruvate Dehydrogenase (PDH) E1 alpha in HH stimulierten Adipozyten.Passend dazu, war die zugehörige PDH Kinase überexprimiert in der zytoplasmatischen Fraktion reifer Fettzellen anzutreffen.Die glykolytischen Schlüsselenzyme Phosphofructokinase (PFK) und Pyruvat Kinase M2 (PKM2) waren ebenfalls signifikant verändert in HH behandelten Proben.Diese Änderungen zeigten einen Glukosefluss in Richtung erhöhter Laktatproduktion.Detailuntersuchungen bestätigten Änderungen der Glykolyse und der mitochondrialen Funktion (TCA Zyklus, OXPHOS) und zeigten auch erhöhte NAD + /NADH und NADP + /NADPH Ratios.Schlussendlich ermittelten diese (phospho)-proteomischen Daten den HH-STW als einen unbekannten Regulator des Energiestoffwechsels, der zu einem erhöhten Glucosedurchsatz und Laktatproduktion führt.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".