PMC

INS 832/13 cells were transfected either with scrambled siRNA [Scr-siRNA] or Rac1-siRNA at a final concentration of 100 nM as indicated in the figure. Following 24 h culture in regular medium, cells were starved overnight and further stimulated with either low [2.5 mM] or high [20 mM] glucose for 30 min. Insulin released into the medium was quantitated by ELISA and expressed as ng/mL [see Methods for experimental details]. Data are mean ± SEM from three independent experiments. * represents p < 0.05 vs. Scr-siRNA transfected low glucose and ** p < 0.05 vs. Scr-siRNA transfected high glucose.

INS 832/13 cells or normal rat islets were treated with either low glucose [CON; 2.5 mM for 6 h]; high glucose [HG; 20 mM for 6 h]; palmitate [PA; 100 μM for 6 h]; cell-permeable C2-CER [30 μM for 6 h] or a mixture of cytokines [CYT; IL-1β, TNFα, IFN-γ = 10 ng/mL each for 24 h] as indicated in the figure. Expression profiles of p47^phox were determined in total lysates by Western blotting [, ]. Please see Methods for experimental details.

Panel A: NSC23766, a selective inhibitor of Tiam1-mediated activation of Rac1, markedly attenuates high glucose-, palmitate-, and C2-CER-induced Rac1 activation in INS 832/13 cells and normal rat islets. INS 832/13 cells or normal rat islets were incubated overnight with either diluent alone or NSC23766 [20 μM] in low serum low glucose media. The cells were then incubated further with either low glucose [CON; 5 mM for 30 min]; high glucose [HG; 20 mM for 30 min]; palmitate [PA; 100 μM for 3 h]; C2-CER [30 μM for 30 min in INS 832/13 cells and 3 h in normal rat islets] in the continuous presence of the diluent or NSC23766. The degree of Rac1 activation was determined by PAK-PBD pull down assay [, ].
Panel B: GGTI-2147, a selective inhibitor of geranylgeranylation of Rac1, markedly attenuates cytomix-induced Rac1 activation in INS 832/13 cells. INS 832/13 cells were incubated overnight with either diluent alone or GGTI-2147 [10 μM] in low serum low glucose media. The cells were then incubated further with either low glucose [CON; 5 mM for 15 min] or a mixture of cytokines [CYT; IL-1β, TNFα, IFN-γ = 10 ng/mL each for 15 min] in the continuous presence of the diluent or GGTI-2147. The degree of Rac1 activation was determined by PAK-PBD pull down assay [, ]. Please see Methods for experimental details.

We propose that exposure of pancreatic β-cells to high glucose, palmitate or cytokines leads to activation of Tiam1/Rac1 signaling axis culminating in the activation of Nox. This, in turn, results in generation of ROS, which adversely affect mitochondrial functions leading to loss in membrane potential thus favoring cytochrome C release into the cytosolic compartment. The latter promotes the activation of pro-apoptotic caspase 3 and associated cleavage of substrate proteins leading to the apoptotic demise of the β-cell. Inhibition of Nox by DPI appears to prevent such metabolic defects. Potential involvement of Taim1/Rac1 signaling cascade was verified by the use of NSC23766, a selective inhibitor of this pathway. It also appears that intracellularly-generated CER via the de novo pathway from palmitate mediates activation of Tiam1/Rac1 and Nox activation steps since fumonisin B1, a selective inhibitor of the ceramide biosynthesis from palmitate largely restored palmitate effects []. Furthermore, C2-CER, a cell permeable analogue of CER, mimicked palmitate effects []. It is likely that exposure of isolated β-cells to glucose also leads to intracellular accumulation of CER via activation of sphingomyelinases endogenous to the β-cell. In addition, glucose can directly activate Tiam1/Rac1 signaling pathway independent of its effects on intracellular CER levels. Our recently published evidence indicates that exposure of isolated β-cells to a mixture of cytokines [i.e., IL-1β, TNFα and IFNγ] also leads to increase in the expression of p47^phox, Tiam1/Rac1 activation and metabolic dysfunction []. Together, these data appear to implicate critical regulatory roles of Tiam1/Rac1/Nox signaling steps in the metabolic dysfunction of the β-cell under the duress of various noxious stimuli.
Abbreviations used in this figure are: SMase: sphingomyelinase; Tiam1: T-lymphoma invasion and metastasis1; DPI: diphenyleneiodonium chloride.