A Novel Assay for Insulin Transcytosis Reveals an Unexpected Role for Clathrin
Bibliographic record
Abstract
Before insulin can reach its target tissues it must cross the micovasculature, a process which has been shown to be rate-limiting in insulin action. Most evidence to date suggests that the major route by which insulin is transported across the endothelium is through individual cells (transcytosis); however, little is known about how this is regulated due to technical limitations. We devised a novel method of studying insulin transcytosis across live human adipose microvascular endothelial cells (HAMECs) using total internal reflection fluorescence microscopy. Using this assay we study the route by which insulin destined for transcytosis is internalized. We show that insulin transcytosis is dynamin-dependent as treatment with Dyngo 4a ablated insulin uptake and transcytosis. Intriguingly, insulin transcytosis was unaffected by cholesterol depletion by methyl-β-cyclodextrin or nystatin. Moreover, caveolin-1 depletion by siRNA had no effect and there was little colocalization of insulin with caveolin-1 by immunofluorescence (IF). Instead, interfering with clathrin-mediated endocytosis with Pitstop 2 or siRNA to clathrin heavy chain significantly impaired transcytosis. Insulin also colocalized with clathrin-heavy chain by IF. Together, these data indicate that insulin transcytosis in HAMECs is clathrin-dependent, in contrast to what has been reported previously for insulin uptake in macrovascular endothelial cells. Elucidating the mechanisms of insulin transcytosis may identify novel targets for insulin resistance.
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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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.003 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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".