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Published on: December 7, 2017
RhoG-Rac1 Signaling Pathway Mediates Metabolic Dysfunction of the Pancreatic Beta-Cells Under Chronic Hyperglycemic
Sri Aneesha Chundru1,2, Ali Harajli1,2, Mirabela Hali1,2
1Biomedical Research Service, John D. Dingell VA Medical Center, Detroit, MI, USA.
Background/Aims:
Published evidence suggests regulatory roles for small G proteins (Cdc42 and Rac1) in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. More recent evidence suggests novel roles for these G proteins, specifically Rac1, in the induction of metabolic dysfunction of the islet beta-cell under the duress of a variety of stress conditions. However, potential upstream regulators of sustained activation of Rac1 have not been identified in the beta-cell. Recent studies in other cell types have identified RhoG, a small G protein, as an upstream regulator of Rac1 under specific experimental conditions. Herein, we examined putative roles for RhoG in islet beta-cell dysregulation induced by glucotoxic conditions.
Methods:
Expression of RhoG or GDIγ was suppressed by siRNA transfection using the DharmaFect1 reagent. Subcellular fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit. The degree of activation of Rac1 was assessed using a pull-down assay kit. Extent of cell death was quantified using a Cell Death Detection ELISAplus kit.
Results:
RhoG is expressed in human islets, rat islets, and clonal INS-1 832/13 cells. siRNA-RhoG markedly attenuated sustained activation of Rac1 and caspase-3 in INS-1 832/13 cells exposed to hyperglycemic conditions (20 mM; 24 hours). In a manner akin to Rac1, which has been shown to translocate to the nuclear fraction to induce beta-cell dysfunction under metabolic stress, a significant increase in the association of RhoG with the nuclear fraction was observed in beta-cells under the duress of metabolic stress. Interestingly, GDIγ, a known regulator of RhoG, remained associated with non-nuclear fraction under conditions RhoG and Rac1 translocated to the membrane. Lastly, siRNA-RhoG modestly attenuated pancreatic beta-cell demise induced by high glucose exposure conditions, but such an effect was not statistically significant.
Conclusion:
Based on these data we conclude that RhoG-Rac1 signaling module plays critical regulatory roles in promoting mitochondrial dysfunction (caspase-3 activation) of the islet beta cell under metabolic stress.
Insights
The small G protein RhoG regulates Rac1 activation and mitochondrial dysfunction in pancreatic beta-cells under high glucose conditions. Suppressing RhoG attenuated Rac1 activation and caspase-3, suggesting a role in metabolic stress.
Area of Science:
- Cell Biology
- Endocrinology
- Molecular Biology
Background:
- Small G proteins like Cdc42 and Rac1 are implicated in insulin secretion and beta-cell dysfunction under stress.
- Upstream regulators of sustained Rac1 activation in beta-cells remain unidentified.
- RhoG, a small G protein, has been identified as a potential upstream regulator of Rac1 in other cell types.
Purpose of the Study:
- To investigate the role of RhoG in islet beta-cell dysregulation induced by glucotoxic conditions.
- To determine if RhoG acts as an upstream regulator of Rac1 in pancreatic beta-cells under metabolic stress.
Main Methods:
- Small interfering RNA (siRNA) was used to suppress RhoG or GDIγ expression.
- Subcellular fractions were isolated to assess protein localization.
- Rac1 activation was measured using a pull-down assay.
- Cell death was quantified using ELISA.
Main Results:
- RhoG is expressed in human and rat islets, as well as INS-1 832/13 cells.
- siRNA-RhoG attenuated Rac1 activation and caspase-3 in beta-cells under hyperglycemic conditions.
- RhoG translocated to the nuclear fraction under metabolic stress, similar to Rac1.
- siRNA-RhoG showed a trend towards attenuating high glucose-induced beta-cell death, but not significantly.
Conclusions:
- The RhoG-Rac1 signaling module is critical in promoting mitochondrial dysfunction, evidenced by caspase-3 activation, in islet beta-cells under metabolic stress.
- RhoG plays a regulatory role in the response of beta-cells to glucotoxicity.
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