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A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Karunanithi S, Xiong T, Uhm M, Leto D, Sun J, Chen XW, Saltiel AR - Mol. Biol. Cell (2014)

Bottom Line: Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2.Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10.Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.

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Rlf is required for maximal glucose uptake in 3T3-L1 adipocytes. (A) siRNA-mediated depletion of indicated genes in 3T3-L1 adipocytes. Six days after knockdown, cells were starved, and 2-deoxyglucose uptake was measured. A representative assay from five independent repetitions is shown. *p < 0.05, **p < 0.01. (B) Knockdown efficiency and insulin signaling in conditions in A. (C) Glut4 translocation assay. 3T3-L1 adipocytes stably expressing Myc-Glut4-eGFP were used for knockdown of indicated genes. Scrambled siRNA was used as control. Immunostaining for Myc (red) in nonpermeabilized cells indicates Glut4 translocation to plasma membrane. GFP levels in cells indicate total Glut4 expressed. A merge of GFP and Myc is shown. Bar, 10 μm. (D) Number of cells that show Myc staining at the rim in the total number of GFP-positive cells was counted for control and Rab10 and Rlf knockdown. Percentage of cells that undergo Glut4 translocation is graphed. Error bars are indicative of at least three independent experiments. *p < 0.05. (E) Knockdown efficiency at protein level is shown by immunoblots. Intact insulin signaling under conditions in D is shown by Western blotting.
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Figure 5: Rlf is required for maximal glucose uptake in 3T3-L1 adipocytes. (A) siRNA-mediated depletion of indicated genes in 3T3-L1 adipocytes. Six days after knockdown, cells were starved, and 2-deoxyglucose uptake was measured. A representative assay from five independent repetitions is shown. *p < 0.05, **p < 0.01. (B) Knockdown efficiency and insulin signaling in conditions in A. (C) Glut4 translocation assay. 3T3-L1 adipocytes stably expressing Myc-Glut4-eGFP were used for knockdown of indicated genes. Scrambled siRNA was used as control. Immunostaining for Myc (red) in nonpermeabilized cells indicates Glut4 translocation to plasma membrane. GFP levels in cells indicate total Glut4 expressed. A merge of GFP and Myc is shown. Bar, 10 μm. (D) Number of cells that show Myc staining at the rim in the total number of GFP-positive cells was counted for control and Rab10 and Rlf knockdown. Percentage of cells that undergo Glut4 translocation is graphed. Error bars are indicative of at least three independent experiments. *p < 0.05. (E) Knockdown efficiency at protein level is shown by immunoblots. Intact insulin signaling under conditions in D is shown by Western blotting.

Mentions: Rab10 and RalA are established components of the insulin regulatory network and are required for Glut4 translocation and glucose uptake in adipocytes (Chen et al., 2007; Sano et al., 2007, 2008). Because Rlf appears to be an intermediate in this activation cascade, we hypothesized that the GEF may also be essential. We depleted Rlf using siRNA in 3T3-L1 adipocytes before assaying 2-deoxyglucose uptake. The expression of Rlf was reduced by ∼70% after incubation with siRNA oligos (Figure 5B). Knockdown of Rlf caused a 15–20% loss in insulin-stimulated glucose uptake, comparable to the effect achieved by knockdown of RalA (Chen et al., 2007) and Rab10 (Figure 5A). Because RalA and Rab10 are required for translocation of Glut4 storage vesicles to the plasma membrane upon insulin stimulation (Chen et al., 2007; Sano et al., 2007), we also tested the effect of Rlf knockdown on Glut4 translocation. Knockdown of Rlf in 3T3-L1 adipocytes stably expressing Myc-Glut4–enhanced green fluorescent protein (eGFP; Bogan et al., 2001; Lodhi et al., 2007) caused a significant reduction (∼25%) in surface Glut4 levels similar to the effect of knockdown of RalA and Rab10 (Figure 5, C–E). Together these data suggest that Rlf is a new component required for Glut4 translocation and is essential for maximal glucose uptake in adipocytes.


A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Karunanithi S, Xiong T, Uhm M, Leto D, Sun J, Chen XW, Saltiel AR - Mol. Biol. Cell (2014)

Rlf is required for maximal glucose uptake in 3T3-L1 adipocytes. (A) siRNA-mediated depletion of indicated genes in 3T3-L1 adipocytes. Six days after knockdown, cells were starved, and 2-deoxyglucose uptake was measured. A representative assay from five independent repetitions is shown. *p < 0.05, **p < 0.01. (B) Knockdown efficiency and insulin signaling in conditions in A. (C) Glut4 translocation assay. 3T3-L1 adipocytes stably expressing Myc-Glut4-eGFP were used for knockdown of indicated genes. Scrambled siRNA was used as control. Immunostaining for Myc (red) in nonpermeabilized cells indicates Glut4 translocation to plasma membrane. GFP levels in cells indicate total Glut4 expressed. A merge of GFP and Myc is shown. Bar, 10 μm. (D) Number of cells that show Myc staining at the rim in the total number of GFP-positive cells was counted for control and Rab10 and Rlf knockdown. Percentage of cells that undergo Glut4 translocation is graphed. Error bars are indicative of at least three independent experiments. *p < 0.05. (E) Knockdown efficiency at protein level is shown by immunoblots. Intact insulin signaling under conditions in D is shown by Western blotting.
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Figure 5: Rlf is required for maximal glucose uptake in 3T3-L1 adipocytes. (A) siRNA-mediated depletion of indicated genes in 3T3-L1 adipocytes. Six days after knockdown, cells were starved, and 2-deoxyglucose uptake was measured. A representative assay from five independent repetitions is shown. *p < 0.05, **p < 0.01. (B) Knockdown efficiency and insulin signaling in conditions in A. (C) Glut4 translocation assay. 3T3-L1 adipocytes stably expressing Myc-Glut4-eGFP were used for knockdown of indicated genes. Scrambled siRNA was used as control. Immunostaining for Myc (red) in nonpermeabilized cells indicates Glut4 translocation to plasma membrane. GFP levels in cells indicate total Glut4 expressed. A merge of GFP and Myc is shown. Bar, 10 μm. (D) Number of cells that show Myc staining at the rim in the total number of GFP-positive cells was counted for control and Rab10 and Rlf knockdown. Percentage of cells that undergo Glut4 translocation is graphed. Error bars are indicative of at least three independent experiments. *p < 0.05. (E) Knockdown efficiency at protein level is shown by immunoblots. Intact insulin signaling under conditions in D is shown by Western blotting.
Mentions: Rab10 and RalA are established components of the insulin regulatory network and are required for Glut4 translocation and glucose uptake in adipocytes (Chen et al., 2007; Sano et al., 2007, 2008). Because Rlf appears to be an intermediate in this activation cascade, we hypothesized that the GEF may also be essential. We depleted Rlf using siRNA in 3T3-L1 adipocytes before assaying 2-deoxyglucose uptake. The expression of Rlf was reduced by ∼70% after incubation with siRNA oligos (Figure 5B). Knockdown of Rlf caused a 15–20% loss in insulin-stimulated glucose uptake, comparable to the effect achieved by knockdown of RalA (Chen et al., 2007) and Rab10 (Figure 5A). Because RalA and Rab10 are required for translocation of Glut4 storage vesicles to the plasma membrane upon insulin stimulation (Chen et al., 2007; Sano et al., 2007), we also tested the effect of Rlf knockdown on Glut4 translocation. Knockdown of Rlf in 3T3-L1 adipocytes stably expressing Myc-Glut4–enhanced green fluorescent protein (eGFP; Bogan et al., 2001; Lodhi et al., 2007) caused a significant reduction (∼25%) in surface Glut4 levels similar to the effect of knockdown of RalA and Rab10 (Figure 5, C–E). Together these data suggest that Rlf is a new component required for Glut4 translocation and is essential for maximal glucose uptake in adipocytes.

Bottom Line: Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2.Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10.Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.

Show MeSH
Related in: MedlinePlus