Dominant negative α-subunit of farnesyl- and geranylgeranyl-transferase I inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes

Abstract

OBJECTIVE:To investigate whether the expression of a dominant negative (DN) farnesyl-and geranygeranyl-transferase I (FTase=GGTase I) a-subunit in 3T3-L1 pre-adipocytes can inhibit insulin's ability to induce differentiation. DESIGN: 3T3-L1 pre-adipocytes were stably transfected with vector alone or vector expressing a mutated DN FTase=GGTase I a-subunit (S60A)(S62A) and incubated in serum-free medium in the absence and presence of insulin. MEASUREMENTS: Various assays were performed to determine the effect of DN FTase=GGTase I a-subunit expression in 3T3-L1 pre-adipocyte on insulin-induced DNA synthesis, cell count, phosphorylation of the FTase=GGTase I a-subunit, FTase and GGTase I activity, amounts of prenylated p21Ras and RhoA, phosphorylation of MAP kinase and Akt, and differentiation to mature fat cells. RESULTS: Expression of DN FTase=GGTase I a-subunit inhibited insulin's ability to increase DNA synthesis, cell count, FTase and GGTase I activity, amounts of prenylated p21Ras and RhoA, and magnitude of phosphorylation of MAP kinase. Expression of DN FTase=GGTase I a-subunit in 3T3-L1 pre-adipocytes was without effect on insulin-induced Akt phosphorylation. CONCLUSION: Expression of DN FTase=GGTase I a-subunit inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes to mature adipocytes, and thus could indicate potential therapeutic avenues to assuage the deleterious effects of obesity and type 2 diabetes.

Dominant-Negative α-Subunit of Farnesyl- and Geranyltransferase Inhibits Glucose-Stimulated, but Not KCl-Stimulated, Insulin Secretion in INS 832/13 Cells

“…They also reported that substitution of alanine for two serine residues (i.e., S60A and S62A) of the ␣-subunit of FTase/GGTase creates a dominant-negative mutant for both PPTases (the one used herein). Finally, these investigators also demonstrated that overexpression of the dn FTase/GGTase ␣-mutant markedly attenuated the ability of insulin to increase the activities of FTase/ GGTase and the abundance of prenylated p21Ras and Rho A (29,30). Taken together, and at least based on the transfection experiments involving a mutant lacking serine phosphorylation sites, these results have led us to conclude that glucose-mediated effects on insulin secretion require phosphorylation of serine-60 and -62 residues of the PPTase ␣-subunits.…”

“…Translocation and membrane association of Rac1 in INS 832/13 cells. This was determined according to a previously described method (29,30). In brief, lysates derived from low-or high-glucose-treated INS 832/13 cells transfected with either the expression vector or the inactive mutant of FTase/GGTase ␣-subunit were subjected to single-step centrifugation at 105,000g for 60 min.…”

“…Subcellular distribution of FTase/GGTase in INS 832/13 cells. Total membrane and soluble fractions were isolated according to a previously described method (29,30). In brief, INS 832/13 cell lysates were subjected to single-step centrifugation at 105,000g for 60 min.…”

Protein prenylation and human diseases: a balance of protein farnesylation and geranylgeranylation

“…It is also reported that insulin can activate the geranylgeranyl transferase and induce Rab geranylgeranylated in 3T3-L1 preadipocytes. Inhibiting the geranylgeranyl transferase activity can down-regulate the phosphorylation of MAPK pathway without affecting the PI3K/AKT pathway in 3T3-L1 preadipocytes [27].…”

Statins in the Treatment of Polycystic Ovary Syndrome

“…Insulin simulates the phosphorylation and activation of farnesyl transferase 122,123,[126][127][128] , thereby augmenting the isoprenylation of Ras and other small GTPases [122][123][124]129 . This leads to cellular proliferation by activation of the MAPK pathway, which is mediated by the small GTPases and their downstream effectors, such as Ras-Raf-extracellular signal-regulated kinase 1/2 (Erk 1/2).…”