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.2009 Oct;30(10):1866-73.
doi: 10.1016/j.peptides.2009.06.021. Epub 2009 Jun 27.

Enterostatin alters protein trafficking to inhibit insulin secretion in Beta-TC6 cells

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Enterostatin alters protein trafficking to inhibit insulin secretion in Beta-TC6 cells

Miejung Park et al. Peptides.2009 Oct.

Abstract

Enterostatin is a peptide that regulates dietary fat intake in rodents and inhibits insulin secretion from pancreatic beta cells. Microarray studies of the genomic response of both a human hepatoma cell line (HepG2 cells) and a mouse hypothalamic cell line (GT1-7 cells) to enterostatin suggested that it might regulate protein trafficking. Using semi-quantitative real-time PCR and Western blot analysis, we confirmed that enterostatin upregulated Scamp2 and down regulated Dynamin2 in these cell lines. The receptor for enterostatin is the F1-ATPase beta subunit. We transfected HepG2 cells with either a green fluorescent protein (GFP) tagged F1-ATPase beta subunit or a red fluorescent protein (RFP) tagged F1-ATPase alpha subunit to study the effects of enterostatin on translocation of its own receptor protein. Enterostatin induced movement of GFP-beta subunit to the cell periphery area but did not have any effect on the localization of RFP-alpha subunit protein in HepG2. As Scamp2 is involved in glucose uptake in mouse Beta-TC6 insulinoma cells we tested enterostatin's effect in Beta-TC6 cells. Glucose stimulated insulin release was inhibited by enterostatin as reported previously. Using siRNA to Scamp2 did not change glucose stimulated insulin release but siRNA to Dynamin2 and dominant negative Dynamin2 (Dyn K44A) inhibited glucose stimulated insulin release and abolished the response to enterostatin. This suggests enterostatin inhibits glucose stimulated insulin release in pancreatic beta cells through down regulation of Dynamin2. This study also suggests that enterostatin might have a more generalized effect on protein trafficking in various cells.

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Figures

Figure 1
Figure 1
F1-ATPase beta subunit-Green Fluorescent Protein (GFP) construct (A) and F1-ATPase alpha subunit-Red Fluorescent Protein (RFP) construct (B).
Figure 2
Figure 2
Effect of enterostatin on gene expression (A) and protein expression (B) of Dynamin2 and Scamp2 in HepG2 cells. Section C shows immunohistochemical images of Scamp2 and Dynamin2 together with nuclei (DAPI staining) of HepG2 cells incubated in presence or absence of enterostatin. All incubations were for one hour. The values shown in panel A are Means ± SEM for 8 observations in each group.
Figure 3
Figure 3
Confocal microscopic images of F1-ATPase beta subunit protein (Green) or alpha subunit protein (Red) in HepG2 cells incubated with (cells A through D; bottom images) or without enterostatin (1μM) (cells A through D; top images) for one hour.
Figure 4
Figure 4
Western blot of F1-ATPase beta subunit in subcellular fractions of HepG2 cells incubated in the presence or absence of enterostatin for 1 hour. 20μg protein from plasma membrane (PM), light mitochondrial fraction (MT) or cytosolic fraction (CYT) was loaded onto the gel. Marker assays showed the MT and PM fractions were free of cross contamination and neither MT or PM marker activity was evident in the cytosolic fraction..
Figure 5
Figure 5
Effects of Dynamin 2 and Scamp 2 on insulin secretion from Beta-TC6 cells. Panel A shows knockdown of Dynamin 2 and Scamp2 by their respective siRNAs (D and S) compared to a control (C) scrambled siRNA. Panel B shows effects of siRNA to Scamp2 and Dynamin 2 on glucose stimulated insulin secretion and on the inhibitory response to enterostatin. Panel C shows that a transfected Dominant Negative Dyn2 (K44A) construct (DN) inhibits insulin secretion and blocks any further inhibitory effect of enterostatin on insulin secretion. Values represent Means ± SEMs for a minimum of 4 observations/group. *** p<0.001 compared to controls or groups as shown.
Figure 6
Figure 6
The lack of effect of a dominant negative Dynamin 2 [DN Dyn2 (K44A)] on the enterostatin stimulation of phospho-PKARIIβ and pERK in Beta TC-6 cells. Wild type (DN −) or dominant negative Dynamin 2 transfected cells (DN +) were incubated 48 hours after transfection with enterostatin (0.01or 1.0μM) for 15 minutes.
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