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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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Rab10 activates RalA. (A) 3T3-L1 adipocytes expressing the indicated active G proteins using lentiviral vectors were subjected to 2-deoxyglucose uptake assay. Glucose uptake under basal conditions are shown. *p < 0.05; **p < 0.01. (B) Glucose uptake under conditions in A upon insulin stimulation. (C) Level of indicated proteins in 3T3-L1 adipocytes under conditions in A and B were assessed by immunoblots. (D) 3T3-L1 adipocytes were electroporated with 50 μg of the indicated plasmids. Two days after electroporation, cells were starved for 16 h and treated with 10 nM insulin for 5 min, and pull-down assay was performed using GST-Sec5-RBD beads. (E) Immunoblots from three independent experiments as in D were quantified using ImageJ (National Institutes of Health, Bethesda, MD) and graphed. *p < 0.05. (F) Indicated plasmids were expressed in Cos-1 cells, and cells were lysed after 24 h. Pull down with GST-RalBP1 beads was performed, and samples were run on SDS–PAGE and immunoblotted. (G) RalA activity as measured by quantification of three independent experiments as in F. Statistical tests were performed by comparing each condition to that of the vector. *p < 0.01.
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Figure 2: Rab10 activates RalA. (A) 3T3-L1 adipocytes expressing the indicated active G proteins using lentiviral vectors were subjected to 2-deoxyglucose uptake assay. Glucose uptake under basal conditions are shown. *p < 0.05; **p < 0.01. (B) Glucose uptake under conditions in A upon insulin stimulation. (C) Level of indicated proteins in 3T3-L1 adipocytes under conditions in A and B were assessed by immunoblots. (D) 3T3-L1 adipocytes were electroporated with 50 μg of the indicated plasmids. Two days after electroporation, cells were starved for 16 h and treated with 10 nM insulin for 5 min, and pull-down assay was performed using GST-Sec5-RBD beads. (E) Immunoblots from three independent experiments as in D were quantified using ImageJ (National Institutes of Health, Bethesda, MD) and graphed. *p < 0.05. (F) Indicated plasmids were expressed in Cos-1 cells, and cells were lysed after 24 h. Pull down with GST-RalBP1 beads was performed, and samples were run on SDS–PAGE and immunoblotted. (G) RalA activity as measured by quantification of three independent experiments as in F. Statistical tests were performed by comparing each condition to that of the vector. *p < 0.01.

Mentions: Recent evidence on the sequential action of G proteins in vesicular trafficking (Mizuno-Yamasaki et al., 2012) suggests that G protein cascades could be a general mechanism in the regulation of vesicle recycling. Of interest, upon insulin stimulation in fat cells, Akt1/2 phosphorylates and inactivates both AS160 (Kane et al., 2002) and the RalGAP complex RGC1/2 (Chen et al., 2011b), leading to the activation of Rab10 and RalA. However, the relative roles of these G proteins and their relationship to each other remain unknown. Whereas the overexpression of constitutively active RalA (G23V/GV) or Rab10 (Q68L/QL) (Figure 2C) independently increased basal glucose uptake in adipocytes (Figure 2A), no additive effects were observed with co-overexpression of the two active G proteins in the same cells. Expression of active RalA modestly increased insulin-stimulated glucose uptake, without further effect from coexpression of Rab10 (QL) (Figure 2B). These data suggest the possibility that RalA and Rab10 might reside in a linear pathway, and we thus determined whether either of the two G proteins might influence the activity of the other. Although overexpression of constitutively active RalA did not alter cellular Rab10 activity, measured as described (Supplemental Figure S2), Rab10 overexpression produced an increase in RalA activity under insulin-stimulated conditions (Figure 2, D and E) as measured by GST-Sec5-RBD pull-down assay. Activation of RalA was dependent on the activation status of Rab10. Overexpression of the constitutively active Rab10 (QL) allele caused hyperactivation of RalA, whereas a GDP-locked version of Rab10 (TN) decreased RalA activity (Figure 2, F and G). These data suggest that Rab10 functions upstream of RalA, and further that its activation can stimulate RalA activity.


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)

Rab10 activates RalA. (A) 3T3-L1 adipocytes expressing the indicated active G proteins using lentiviral vectors were subjected to 2-deoxyglucose uptake assay. Glucose uptake under basal conditions are shown. *p < 0.05; **p < 0.01. (B) Glucose uptake under conditions in A upon insulin stimulation. (C) Level of indicated proteins in 3T3-L1 adipocytes under conditions in A and B were assessed by immunoblots. (D) 3T3-L1 adipocytes were electroporated with 50 μg of the indicated plasmids. Two days after electroporation, cells were starved for 16 h and treated with 10 nM insulin for 5 min, and pull-down assay was performed using GST-Sec5-RBD beads. (E) Immunoblots from three independent experiments as in D were quantified using ImageJ (National Institutes of Health, Bethesda, MD) and graphed. *p < 0.05. (F) Indicated plasmids were expressed in Cos-1 cells, and cells were lysed after 24 h. Pull down with GST-RalBP1 beads was performed, and samples were run on SDS–PAGE and immunoblotted. (G) RalA activity as measured by quantification of three independent experiments as in F. Statistical tests were performed by comparing each condition to that of the vector. *p < 0.01.
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Figure 2: Rab10 activates RalA. (A) 3T3-L1 adipocytes expressing the indicated active G proteins using lentiviral vectors were subjected to 2-deoxyglucose uptake assay. Glucose uptake under basal conditions are shown. *p < 0.05; **p < 0.01. (B) Glucose uptake under conditions in A upon insulin stimulation. (C) Level of indicated proteins in 3T3-L1 adipocytes under conditions in A and B were assessed by immunoblots. (D) 3T3-L1 adipocytes were electroporated with 50 μg of the indicated plasmids. Two days after electroporation, cells were starved for 16 h and treated with 10 nM insulin for 5 min, and pull-down assay was performed using GST-Sec5-RBD beads. (E) Immunoblots from three independent experiments as in D were quantified using ImageJ (National Institutes of Health, Bethesda, MD) and graphed. *p < 0.05. (F) Indicated plasmids were expressed in Cos-1 cells, and cells were lysed after 24 h. Pull down with GST-RalBP1 beads was performed, and samples were run on SDS–PAGE and immunoblotted. (G) RalA activity as measured by quantification of three independent experiments as in F. Statistical tests were performed by comparing each condition to that of the vector. *p < 0.01.
Mentions: Recent evidence on the sequential action of G proteins in vesicular trafficking (Mizuno-Yamasaki et al., 2012) suggests that G protein cascades could be a general mechanism in the regulation of vesicle recycling. Of interest, upon insulin stimulation in fat cells, Akt1/2 phosphorylates and inactivates both AS160 (Kane et al., 2002) and the RalGAP complex RGC1/2 (Chen et al., 2011b), leading to the activation of Rab10 and RalA. However, the relative roles of these G proteins and their relationship to each other remain unknown. Whereas the overexpression of constitutively active RalA (G23V/GV) or Rab10 (Q68L/QL) (Figure 2C) independently increased basal glucose uptake in adipocytes (Figure 2A), no additive effects were observed with co-overexpression of the two active G proteins in the same cells. Expression of active RalA modestly increased insulin-stimulated glucose uptake, without further effect from coexpression of Rab10 (QL) (Figure 2B). These data suggest the possibility that RalA and Rab10 might reside in a linear pathway, and we thus determined whether either of the two G proteins might influence the activity of the other. Although overexpression of constitutively active RalA did not alter cellular Rab10 activity, measured as described (Supplemental Figure S2), Rab10 overexpression produced an increase in RalA activity under insulin-stimulated conditions (Figure 2, D and E) as measured by GST-Sec5-RBD pull-down assay. Activation of RalA was dependent on the activation status of Rab10. Overexpression of the constitutively active Rab10 (QL) allele caused hyperactivation of RalA, whereas a GDP-locked version of Rab10 (TN) decreased RalA activity (Figure 2, F and G). These data suggest that Rab10 functions upstream of RalA, and further that its activation can stimulate RalA activity.

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