Fig 1: Glucagon and Stmn2 are present within secretory granules of pancreatic a-cells. (A) Immunogold labels for both glucagon (10 nm; white arrows) and Stmn2 (18 nm; black arrows) are localized within secretory granules. The low magnification image (25,000×, scale bar = 1 µm) shows the ultrastructure of one alpha cell. N (nucleus); M (mitochondria); ER (endoplasmic reticulum); SG (secretory granule). The magnified image (41,000×, scale bar = 0.6 µm) highlights the presence of immunogold labels within a single secretory granule. (B) Isolated mouse islets were incubated in the presence or absence of 25 mM Arg for 20 min. Both glucagon and Stmn2 secretion from murine islets is stimulated by arginine. Values were normalized to the non-stimulated condition and expressed as mean ± SEM (n = 5–6). *p < 0.01.
Fig 2: Proposed pathways by which stathmin-2 modulates glucagon secretion from aTC1-6 cells. The model denotes status of glucagon secretion from aTC1-6 cells in normal physiology or in the event of Stmn2- depletion or overexpression. Blue arrows indicate the normal trafficking of glucagon, together with Stmn2, to secretory granules, where they are stored until their release is triggered by a stimulus. Stmn2 overexpression (red arrows) reduces the amount of glucagon available for secretion by diverting secretory granules to lysosomes. Stmn2 depletion (black arrows) reduces the trafficking of glucagon into secretory granules and promotes the constitutive release of glucagon.
Fig 3: The presence of stathmin-2 and glucagon within secretory granules of α-cells, and parallel alterations in secretion of glucagon and Stmn2 from α-cells in both non-diabetic and diabetic mice. (A) Double immunogold labeling transmission electron microscopy of islets in non-diabetic and (B) STZ-induced diabetic mice was for glucagon (10 nm gold, white arrows in the magnified section) and stathmin-2 (18 nm gold, black arrows in the magnified section). The low magnification images (25000×, scale bar = 1 μm) show the ultrastructure of one α-cell. N (nucleus); ER (endoplasmic reticulum); SG (secretory granule); PM (plasma membrane). The magnified images (41000×, scale bar = 0.6 μm) highlight the presence of immunogold labels within a single secretory granule. (C) Secretion of glucagon and islet glucagon content in isolated islets of non-diabetic (C; n = 7) and diabetic mice (STZ; n = 7) at the presence or absence of Arginine (25 mM, 20 min). Secretion values were normalized by baseline control secretion and expressed as fold changes. Glucagon contents were normalized by baseline control and expressed as percent changes. (D) Secretion of Stmn2 and islet Stmn2 content in isolated islets of non-diabetic (C; n = 7) and diabetic (STZ; n = 7) mice in the presence or absence of Arginine (25 mM, 20 min). Secretion values were normalized by baseline secretion in control and expressed as fold changes. Glucagon content was normalized by baseline content in control and expressed as percent changes. Values were expressed as mean ± SEM and compared among groups by 1-Way ANOVA at α = 0.05. *p < .05; **p < .01; ***p < .001
Fig 4: Altered ratios of stathmin-2 and glucagon in islets of STZ-induced diabetic mice. Islets were immunostained for stathmin-2 (Stmn2) and glucagon and images were acquired as described in Methods. (A) Expression of glucagon and Stmn2 were determined in islets of non-diabetic and diabetic mice by immunofluorescence intensity analysis. (B) Ratios of glucagon: Stmn2 levels were calculated per µm2 of islets in non-diabetic and diabetic mice. (C) Linear regression analysis on binary image intensities of the Stmn2 and glucagon. Filled circles and open squares demonstrate values in non-diabetic and diabetic islets, respectively. (D) Expression of Stmn2 and Gcg mRNA levels were determined in islets of non-diabetic (n = 4) and diabetic (n = 4) mice by qRT-PCR. Gene expression levels were normalized to that of 18S rRNA. For each gene, alterations in the diabetic condition were normalized by the corresponding control group and expressed as percent of matched control. Comparison between control and STZ groups was done by t-test, α = 0.05
Fig 5: Stathmin-2 localizes to secretory granules in aTC1-6 cells. aTC1-6 cells were immunostained using primary antibodies against glucagon (GCG, green) and stathmin-2 (Stmn2, red). DAPI (blue) indicates the nucleus in the merged image. Resolution of the images was extended by applying Nyquist XY scan and then 2D- Deconvolution in NIS Elements image analysis software. Images are representative of four biological replicates with 3 technical replicates each. (A) Areas of yellow in the merged image show colocalization of glucagon and Stmn2. (B) Linear regression analysis of binary intensities of glucagon and Stmn2 predicts a significant (p < 0.001) correlation. Each value represents mean intensities of 5-7 cells. The secretion of both glucagon and Stmn2 (C) was significantly increased after KCl stimulation (KCl Stim)for 15 min. (D) Cell Stmn2 and glucagon levels show reduction following KCl stimulation (KCl Stim). Values are expressed as mean ± SEM (n = 5). *p < 0.05. Stmn2 colocalizes with the secretory granule proteins ChgA (E) and VAMP2 (F), as indicated by yellow punctate staining. (G) The extent of colocalization was analyzed by Pearson correlation coefficient for Stmn2 with ChgA or with VAMP2.
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