Journal: bioRxiv

Article Title: Metabolic STAMP for deciphering GPCR-regulated insulin secretion by pancreatic β cells

doi: 10.1101/2025.10.03.680349

Figure Lengend Snippet: ( A ) Murine β cells or human islet cells were treated by high glucose alone or together with GPCR agonists to stimulate insulin secretion. During secretion, the following time-controlled samples were collected for metabolic STAMP: cell media, cell lysates, live cells, and fixed cells. (B) Cell medium was analyzed for hormone secretion levels like ELISA and/or bioluminescence based high-throughput assays. (C) Cell lysates were processed for DDA-PASEF phosphoproteomics to map phosphorylation sites of GPCR signaling pathways. Putative kinases were nominated based on in silico analysis and then validated using kinase inhibitor assays. ( D ) Paired live and fixed cell analysis via confocal microscopy were used to assess context specific changes in the following cellular compartments: microtubule cytoskeleton, mitochondria, Golgi and endosomal traffic, and primary cilia.

Article Snippet: Cells were stained with the following live cell imaging dyes: SiR-Tubulin (100nM) for microtubule cytoskeleton (Cytoskeleton Inc.; Cat #: CY-SC002), BODIPY FL C5-Ceramide (5μM) for Golgi and plasma membrane (Thermofisher; Cat #: D3521), and Mitotracker Red CMXRos (200nM) for mitochondria (Thermofisher; Cat #: M7512).

Techniques: Enzyme-linked Immunosorbent Assay, High Throughput Screening Assay, Data-dependent acquisition, Phospho-proteomics, Protein-Protein interactions, In Silico, Cell Analysis, Confocal Microscopy

Journal: bioRxiv

Article Title: Metabolic STAMP for deciphering GPCR-regulated insulin secretion by pancreatic β cells

doi: 10.1101/2025.10.03.680349

Figure Lengend Snippet: ( A ) Normalized relative insulin secretion levels were determined via luciferase assay was measured in mouse pancreatic β cell line MIN6 either at low glucose (2.8mM) or treatment with combinations of high glucose (17.8mM), TUG891 (100μM), or Exendin4 (20nM). Cells were serum and glucose starved for 1hr before treatment, and samples were collected at 30 min after treatment. N=3. Error bars= SEM. Statistical significance was assessed via One-Way ANOVA, with * P = 0.05; ** P = 0.01; **** P = 0.00001. Plots were generated using GraphPad Prism software. (B-F) UMAP analysis reveals the differential effects of glucose and GPCR stimulation (FFAR4 vs GLP1-R) upon the cellular phosphoproteomic landscape. Size of circle indicates log2 fold change, color of circle represents the known subcellular compartment where that protein functions. The first panel indicates the baseline of all the possible phosphorylations detected, while the subsequent panels show increased circle sizes for the phosphorylations detected that show marked changes in abundance in that stimulation condition. ( G ) Phosphorylated proteins are known to be important for the integrity and function of multiple subcellular compartments. These include cilia and centriole (blue), microtubule and kinesins (orange), Golgi and ER (green), secretory machinery (yellow). In addition, some proteins were previously associated with regulatory machinery of GPCR signaling (navy) and glucose metabolism (grey). Example phosphorylations are shown here in a model figure of a β cell, with key cellular compartments marked. Arrowheads indicate whether it is an increase or a decrease in abundance and which GPCRs/stimulations induce change (black: GLP1-R; white: FFAR4). ( H ) Examples of changes in abundance of pathway-specific phosphorylations during GSIS. Differences are reported as log2 fold change compared to low glucose conditions, and the putative kinase based on the Cantley algorithm are listed for each phosphorylation.

Article Snippet: Cells were stained with the following live cell imaging dyes: SiR-Tubulin (100nM) for microtubule cytoskeleton (Cytoskeleton Inc.; Cat #: CY-SC002), BODIPY FL C5-Ceramide (5μM) for Golgi and plasma membrane (Thermofisher; Cat #: D3521), and Mitotracker Red CMXRos (200nM) for mitochondria (Thermofisher; Cat #: M7512).

Techniques: Luciferase, Generated, Software, Phospho-proteomics

Journal: bioRxiv

Article Title: Metabolic STAMP for deciphering GPCR-regulated insulin secretion by pancreatic β cells

doi: 10.1101/2025.10.03.680349

Figure Lengend Snippet: Changes in key cellular compartments with different stimulation conditions were assessed via IF and imaged via confocal microscopy at 60x magnification. Samples were collected at 5min and 20min after stimulation. The following compartments were assessed, with representative images and image quantification shown for each: (A) Golgi and vesicular traffic machinery morphology and spreading with dotted lines indicated cell borders (magenta: β-COP; cyan: GM130; blue: DAPI) (scale = 10µm), and quantification of mean gray value cytoplasmic β-COP was assessed for each condition (far right); ( B ) mitochondria-Golgi inter-organellar contacts, with dotted lines outlining the Golgi border to reflect whether mitochondrial are excluded (left, low glucose) or overlapping with Golgi (right, high glucose 5min) (blue: DAPI; green: β-COP; red: Complex-I)(scale = 10μm); quantification on left showing what percentage of cells in each condition show evidence of overlap with Golgi, with error bar= SEM( C ) mitochondrial morphology, with blown up insets showing representative regions highlighting how tubular/fragmented the mitochondria are in that condition (green: Complex 1) (scale = 10µm), with average mitochondrial volume being assessed for each condition (below); ( D ) ciliary lengthening (red: acetylated tubulin; blue: DAPI) (scale = 20µm), with graph quantifying average length of cilia in each condition; ( E ) total microtubule density (green: β-tubulin) versus acetylated microtubules and yellow lines marking cell borders (white: acetylated tubulin; blue: DAPI) (scale = 10µm); differences in density were measured as % cell volume encompassed by total tubulin (upper right) versus acetylated tubulin (lower right); Experiments were performed in biological replicate, with ∼100 cells per replicate except for mitochondrial volume which was >2000 mitochondria per condition and ∼20 cells per condition for β-COP. Error bars= SEM. Statistical significance was assessed via One-Way ANOVA, with * P = 0.05; ** P = 0.01; **** P = 0.0001. Plots were generated using GraphPad Prism software.

Article Snippet: Cells were stained with the following live cell imaging dyes: SiR-Tubulin (100nM) for microtubule cytoskeleton (Cytoskeleton Inc.; Cat #: CY-SC002), BODIPY FL C5-Ceramide (5μM) for Golgi and plasma membrane (Thermofisher; Cat #: D3521), and Mitotracker Red CMXRos (200nM) for mitochondria (Thermofisher; Cat #: M7512).

Techniques: Confocal Microscopy, Generated, Software

Journal: bioRxiv

Article Title: Metabolic STAMP for deciphering GPCR-regulated insulin secretion by pancreatic β cells

doi: 10.1101/2025.10.03.680349

Figure Lengend Snippet: (A) Table showing representative phosphorylations detected from human pancreatic islet (3,000 IEQ per condition). Font color indicates which GSIS phosphorylations are shared between human cadaveric islets and mouse MIN6 stable cell lines (pink= MIN6 only, black= human islet only, blue= shared). Phosphorylations are sorted by row based on known cellular compartment and sorted by column based on predicted regulatory kinase (along with the kinase motif analysis below). Dots next to the phosphorylation reference the binary code plot on the right, which highlights the differential abundance depending on experimental conditions. (B) Representative live cell imaging panels of human cadaveric islets showing changes in ciliary length (left) and microtubule dynamics (right) upon different stimulation conditions. Images were collected via confocal timelapse imaging at 40x magnification, collecting z-stacks every ∼5min using far red SiR-Tubulin live cell imaging dye. Scale bar= 10µm.

Article Snippet: Cells were stained with the following live cell imaging dyes: SiR-Tubulin (100nM) for microtubule cytoskeleton (Cytoskeleton Inc.; Cat #: CY-SC002), BODIPY FL C5-Ceramide (5μM) for Golgi and plasma membrane (Thermofisher; Cat #: D3521), and Mitotracker Red CMXRos (200nM) for mitochondria (Thermofisher; Cat #: M7512).

Techniques: Stable Transfection, Phospho-proteomics, Live Cell Imaging, Imaging