Activation and Translocation of Rho (and ADP Ribosylation Factor) by Insulin in Rat Adipocytes: APPARENT INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE

Purushotham Karnam,Mary L Standaert,Lamar Galloway,Robert V Farese

doi:10.1074/jbc.272.10.6136

Purushotham Karnam, Mary L Standaert + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.10.6136

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Abstract

Insulin reportedly (Standaert, M. L., Avignon, A., Yamada, K., Bandyopadhyay, G., and Farese, R. V. (1996) Biochem. J. 313, 1039-1046) activates phospholipase D (PLD)-dependent hydrolysis of phosphatidylcholine (PC) in plasma membranes of rat adipocytes by a mechanism that may involve wortmannin-sensitive phosphatidylinositol (PI) 3-kinase. Because Rho and ADP ribosylation factor (ARF) activate PC-PLD, we questioned whether these small G-proteins are regulated by insulin and PI 3-kinase. We found that insulin provoked a rapid translocation of both Rho and ARF to the plasma membrane and increased GTP loading of Rho. Wortmannin and LY294002 inhibited Rho translocation in intact adipocytes, and the polyphosphoinositide, PI 4,5-(PO4)2, stimulated Rho translocation in adipocyte homogenates. On the other hand, wortmannin did not block GTP loading of Rho. Guanosine 5'-3-O-(thio)triphosphate stimulated both Rho and ARF translocation and activated PC-PLD in homogenates. C3 transferase, which inhibits and depletes Rho, inhibited PC-PLD activation by insulin in intact adipocytes. C3 transferase also inhibited insulin stimulation of [3H]2-deoxyglucose uptake. Our findings suggest that: (a) insulin translocates Rho by a PI 3-kinase-dependent mechanism, but another factor is responsible for GTP loading of Rho; (b) both Rho and ARF may contribute to PC-PLD activation during insulin action; and (c) Rho may be required for insulin stimulation of glucose transport.

Highlights

Dylcholine (PC) is a major signaling system for agonists that activate tyrosine kinases
The present findings suggested that Rho and ADP ribosylation factor (ARF) may participate in the activation of PC-phospholipase D (PLD) by insulin in rat adipocytes
Both Rho and ARF translocated to plasma membranes sufficiently rapidly to contribute to the rapid activation of plasma membrane PC-PLD by insulin, and both G-proteins have been reported to activate PC-PLD (10 –15)

Summary

EXPERIMENTAL PROCEDURES

Adipocytes were prepared from epididymal fat pads (see Ref. 1), equilibrated in glucose-free Krebs Ringer phosphate (KRP) buffer containing 1% bovine serum albumin (BSA), and treated with wortmannin (Sigma), LY294002 (BioMol), and/or insulin (Elanco) as described in the text. To study Rho/ARF translocation in vitro, post-nuclear homogenates were prepared in Buffer I containing 1 mM EDTA and incubated first for 20 min at room temperature to release GDP and for 20 min at 37 °C after adding 10 mM MgCl2 with or without GTP␥S (Sigma) or PI 4,5-(PO4) (PIP2; Fluka). For in vitro assays, labeled cells were sonicated in Buffer I containing 25 mM HEPES (pH 7.4), 1 mM EDTA, 100 mM KCl, and 3 mM NaCl. After equilibration of homogenates for 20 min at room temperature to release GDP, 1 ␮M CaCl2, 5 mM MgCl2, 2.5% ethanol, and GTP␥S were added, and incubation at 37 °C was continued for 30 min. Cells were electroporated in the absence or the presence of C3 transferase and incubated for designated times prior to assessment of basal and insulin-stimulated 2-DOG uptake

RESULTS

DISCUSSION

Plasma membrane