c-Crk, a Substrate of the Insulin-like Growth Factor-1 Receptor Tyrosine Kinase, Functions as an Early Signal Mediator in the Adipocyte Differentiation Process

Abstract

Differentiation of 3T3-L1 preadipocytes into adipocytes is induced by a combination of inducers, including a glucocorticoid, an agent that elevates cellular cAMP, and a ligand of the insulin-like growth factor-1 receptor. Previous studies have implicated protein-tyrosine phosphatase (PTPase) HA2, a homologue of PTPase 1B, in the signaling cascade initiated by the differentiation inducers. Vanadate, a potent PTPase inhibitor, blocks adipocyte differentiation at an early stage in the program, but has no effect on the mitotic clonal expansion required for differentiation. Exposure of preadipocytes to vanadate along with the inducing agents led to the accumulation of pp35, a phosphotyrosyl protein that is a substrate for PTPase HA2. pp35 was purified to homogeneity and shown by amino acid sequence and mass analyses of tryptic peptides to be c-Crk, a known cytoplasmic target of the insulin-like growth factor-1 receptor tyrosine kinase. Transfection of 3T3-L1 preadipocytes with a c-Crk antisense RNA expression vector markedly reduced c-Crk levels and prevented differentiation into adipocytes. Studies with C3G, a protein that binds to the SH3 domain in c-Crk, showed that phosphorylation of c-Crk rendered the SH3 domain inaccessible to C3G. Taken together, these findings indicate that locking c-Crk in the phosphorylated state with vanadate prevents its participation in the signaling system that initiates adipocyte differentiation.Adipocytes serve an important function in the energy economy of higher organisms, providing a large energy reserve that can be mobilized when needed. Thus, when caloric intake exceeds expenditure, metabolite flux is diverted into triglyceride synthesis for storage in adipocytes. Conversely, when caloric expenditure exceeds intake, this triglyceride reserve is mobilized as free fatty acids to provide physiological fuel for use by other tissue/cell types. The need for an energy reserve begins at birth when the newborn must be prepared to survive periods of energy deprivation. Adipocytes, which provide this reserve, develop late in embryonic life, with major expansion of this cell population occurring after birth. The adipose lineage arises from the same multipotent stem cells of mesodermal origin that give rise to the muscle and cartilage lineages.Established preadipocyte cell lines, e.g. the 3T3-L1 preadipocytes, which can be induced to differentiate into adipocytes in cell culture, provide faithful models with which to investigate the adipocyte differentiation program (1-6). When exposed to the appropriate differentiation inducers, including IGF-1 1 (or insulin at a non-physiologically high concentration), dexamethasone (a glucocorticoid), and 1-methyl-3-isobutylxanthine (MIX; a cAMP phosphodiesterase inhibitor that increases intracellular cAMP), 3T3-L1 preadipocytes differentiate into cells that express the adipocyte phenotype (6). Induction of the adipocyte differentiation program involves at least three different signal transduction systems, including those mediated by the glucocorticoid rec...

Krüppel-like factor KLF9 regulates PPARγ transactivation at the middle stage of adipogenesis

“…The 2-day post-confluent cells were induced for adipocyte differentiation following the protocol described previously. 42 Cytoplasmic triglyceride droplets were visible by day 4. The differentiated 3T3-L1 adipocyte monolayer was stained with Oil-red-O.…”

“…The differentiated 3T3-L1 adipocyte monolayer was stained with Oil-red-O. 42 The Oilred-O in triglyceride droplets was extracted with 100% isopropanol and OD 510 nm was determined.…”

Protein Kinase B/AKT 1 Plays a Pivotal Role in Insulin-like Growth Factor-1 Receptor Signaling Induced 3T3-L1 Adipocyte Differentiation

“…Cell Culture, Differentiation Induction of 3T3-L1 Preadipocytes, and Oil Red-O Staining-3T3-L1 preadipocytes were cultured and induced to differentiate following the protocol described previously (23)(24)(25)(26). Two-day post-confluent 3T3-L1 preadipocytes and RNAi cells were induced with standard differentiation induction medium containing 1-methyl-3-isobutylxanthine (MIX), dexamethasone (DEX), and insulin for 2 days, followed by medium supplemented with insulin for additional 2 days.…”

Crk family adaptors–signalling complex formation and biological roles

“…Since both of these IRS proteins apparantly have some role in regulating cell proliferation, such complexes may act in concert with other well known binders of IRS proteins like PI3 kinase, SHP2 and Grb2 to generate appropriate signals downstream from insulin, IGF-1, PDGF and other growth factors in a speci®c cell context (Sorokin et al, 1998;Nakashima et al, 1999;Qu et al, 1999). Other roles, for example as a signal mediator in adipocyte di erentiation, have also been recently suggested (Jin et al, 2000). Sorokin and Reed have reported a switch of Crk from p130Cas to which it is initially bound onto the IRS-1 protein in response to insulin stimulation (Sorokin and Reed, 1998).…”