Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

Abstract

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time-and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by -80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G-and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet 3-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane. INTRODUCTIONThe actin filaments (AFs)1 are among the components of the cytoskeleton that, in addition to maintaining cell shape, participate in contraction, mitosis, migration, endocytosis, and secretion (Korn, 1982;Howell and Tyhurst, 1986;Pollard and Cooper, 1986;Stossel, 1989;Schelling et al., 1992;Burgoyne and Morgan, 1993). AFs are formed by polymerization of G-actin to F-actin (Korn, 1982;Pollard and Cooper, 1986). The extent of actin polymerization is controlled by the free G-actin concentration, the actin-binding proteins, and co...

Forkhead box O1 mediates defects in palmitate-induced insulin granule exocytosis by downregulation of calcium/calmodulin-dependent serine protein kinase expression in INS-1 cells

“…Knockdown of CASK attenuated the anchoring of insulin granules to cell membranes In vitro, the SH3 domain of CASK combines with actin-binding protein 4.1, which binds with F-actin, a contractile protein involved in nucleation and aggregation, and links the extracellular matrix with the intracellular cytoskeleton [26,27]. To determine whether the effect of CASK on insulin secretion was associated with F-actin, we used FITC green fluorescent-labelled CASK and tetramethylrhodamine isothiocyanate (TRITC)-phalloidin fluorescent-labelled F-actin.…”

“…F-actin, a cytoskeleton protein, prevents the anchoring and fusion of insulin vesicles in the plasma membrane [26,27]. It can combine with CASK-SH3 through protein 4.1 in the haemocyte [24].…”

Regulated von Willebrand Factor Secretion Is Associated With Agonist-Specific Patterns of Cytoskeletal Remodeling in Cultured Endothelial Cells

“…These observations suggest a mechanistic link between these 2 events, as has been suggested in other cell types. 26,27 However, preventing stress fiber formation by CCE pretreatment potentiated thrombin-induced vWF release. Pretreatment with the more specific C3-C2IN toxin prevented stress fiber formation and potentiated vWF release in response to thrombin, histamine, and A23187 but not to cAMP-raising agents.…”

Small GTP-binding Protein TC10 Differentially Regulates Two Distinct Populations of Filamentous Actin in 3T3L1 Adipocytes

“…Furthermore, in some cases disruption of the actin cytoskeleton markedly potentiates agonist-stimulated secretion ( Lelkes et al, 1986;Sontag et al, 1988;Matter et al, 1989;Muallem et al, 1995). In contrast however, in many cell systems depletion of F-actin structures either by sequestering actin monomers or by stimulation of actin severing does not stimulate exocytosis but rather results in an inhibition of agonist-induced secretion (Morita et al, 1988;O'Konski and Pandol, 1990;O'Konski and Pandol, 1993;Li et al, 1994). These findings are consistent with a requirement for active actin remodeling during the transport process rather than a requirement for static actin structures.…”