Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin

Abstract

The phosphorylation of a previously uncharacterized protein of apparent Mr Ϸ 140,000 was found to be increased when rat adipocytes were incubated with insulin. The sequences of peptides generated by digesting the protein with trypsin matched perfectly with sequences in mouse lipin. Lipin is the product of the gene that is mutated in fatty liver dystrophy ( fld) mice [Peterfy, M., Phan, J., Xu, P. & Reue, K (2001) Nat. Genet. 27, 121-124], which exhibit several phenotypic abnormalities including hyperlipidemia, defects in adipocyte differentiation, impaired glucose tolerance, and slow growth. When immunoblots were prepared with lipin antibodies, both endogenous adipocyte lipin and recombinant lipin overexpressed in HEK293 cells appeared as bands ranging in apparent Mr from 120,000 to 140,000. Incubating adipocytes with insulin decreased the electrophoretic mobility and stimulated the phosphorylation of both Ser and Thr residues in lipin. The effects of insulin were abolished by inhibitors of phosphatidylinositol 3-OH kinase, and by rapamycin, a specific inhibitor of the mammalian target of rapamcyin (mTOR). The inhibition by rapamycin was blocked by FK506, which competitively inhibits those effects of rapamycin that are mediated by inhibition of mTOR. Moreover, amino acids, which activate mTOR, mimicked insulin by increasing lipin phosphorylation in a rapamycin-sensitive manner. Thus, lipin represents a target of the mTOR pathway, and potentially links this nutrient-sensing pathway to adipocyte development.

Insulin Controls Subcellular Localization and Multisite Phosphorylation of the Phosphatidic Acid Phosphatase, Lipin 1

“…The phosphorylation of lipin was first observed in rat adipocytes, where it was shown to be increased in a rapamycin-sensitive manner in response to insulin (5). The present results demonstrate that insulin-stimulated phosphorylation of lipin occurs in 3T3-L1 adipocytes (Figs.…”

“…Inhibiting PI 3 kinase, which signals upstream of mTOR (30), with wortmannin also blocked the effects of insulin. These results indicate that lipin phosphorylation is controlled by insulin in a similar manner in 3T3-L1 adipocytes and in primary rat adipocytes, where the hormone was previously shown to increase lipin phosphorylation (5).…”

“…Antibodies-The two lipin antibodies, LAb-1 and LAb-2, prepared by immunizing rabbits with peptide conjugates have been described previously (5). The peptide for LAb-1 corresponded to the sequence between Ser 409 and Gly 430 in mouse lipin 1b, and that for LAb-2 corresponded to the last 11 amino acids in the protein.…”

“…The proteins encoded by Lpin1 are expressed in a wide variety of tissues, with the highest levels found in adipose tissue (4,5). Lipin 1b increases markedly as fat cells differentiate, and it is the prevalent isoform in mature adipocytes (9).…”

“…Thus, fld/fld mice express neither lipin 1a nor lipin 1b, a slightly larger protein, which arises from alternative mRNA splicing (4,5). Within a few days of birth fld/fld mice develop hypertriglyceridemia and hepatic steatosis (6).…”