Abstract
Insulin and insulin-like growth factor-I (IGF-I) receptors are highly homologous tyrosine kinase receptors that share many common steps in their signaling pathways and have ligands that can bind to either receptor with differing affinities. To define precisely the signaling specific to the insulin receptor (IR) or the IGF-I receptor, we have generated brown preadipocyte cell lines that lack either receptor (insulin receptor knockout (IRKO) or insulin-like growth factor receptor knockout (IGFRKO)). Control preadipocytes expressed fewer insulin receptors than IGF-I receptors (20,000 versus 60,000), but during differentiation, insulin receptor levels increased so that mature adipocytes expressed slightly more insulin receptors than IGF-I receptors (120,000 versus 100,000). In these cells, insulin stimulated IR homodimer phosphorylation, whereas IGF-I activated both IGF-I receptor homodimers and hybrid receptors. Insulin-stimulated IRS-1 phosphorylation was significantly impaired in IRKO cells but was surprisingly elevated in IGFRKO cells. IRS-2 phosphorylation was unchanged in either cell line upon insulin stimulation. IGF-I-dependent phosphorylation of IRS-1 and IRS-2 was ablated in IGFRKO cells but not in IRKO cells. In control cells, both insulin and IGF-I produced a dose-dependent increase in phosphorylated Akt and MAPK, although IGF-I elicited a stronger response at an equivalent dose. In IRKO cells, the insulin-dependent increase in phospho-Akt was completely abolished at the lowest dose and reached only 20% of the control stimulation at 10 nm. Most interestingly, the response to IGF-I was also impaired at low doses, suggesting that IR is required for both insulin- and IGF-I-dependent phosphorylation of Akt. Most surprisingly, insulin- or IGF-I-dependent phosphorylation of MAPK was unaltered in either receptor-deficient cell line. Taken together, these results indicate that the insulin and IGF-I receptors contribute distinct signals to common downstream components in response to both insulin and IGF-I.
Highlights
Insulin and insulin-like growth factors (IGF-I and IGF-II)1 lead to an assortment of biological effects in insulin target cells
Insulin and insulin-like growth factor-I (IGF-I) Receptors Are Required for Brown Adipocyte Differentiation—To determine the role of insulin receptor (IR) and IGFR in adipogenesis, we have examined the differentiation capacity of brown preadipocyte cell lines that lack or overexpress each of these receptors created by homologous recombinant gene targeting [27, 28]
We have shown that the insulin receptor that lacks exon 11 (IRA) is the predominant isoform expressed in brown preadipocytes [29], and we have used this isoform for the following overexpression studies
Summary
Materials—Antibodies used for immunoprecipitation and immunoblotting included anti-insulin receptor  subunit (C-19), anti-IGFR  subunit (C-20), anti-IGFR ␣ subunit (N-20), anti-IRS-1 (C-20), and anti-peroxisome proliferator-activated receptor ␥ (E-8) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-IRS-2 and anti-phosphotyrosine 4G10 (kindly provided by Morris White, Joslin Diabetes Center, Boston, MA); anti-insulin receptor C terminus (Joslin Diabetes Center, Boston, MA); anti-phospho-Akt and anti-phospho-MAPK (New England Biolabs, Beverly, MA); and anti-Glut (Chemicon International Inc., Temecula, CA). Cell Isolation and Culture—Cells that were homozygous for a floxed exon 4 of the insulin receptor (IRlox) were used as controls for all studies. Adipocyte Differentiation and Oil Red O Staining—To differentiate cells, preadipocytes were first grown to confluence in culture medium containing 20 nM insulin and 1 nM triiodothyronine (differentiation medium). Plasmids and Retroviral Infection—Generation of IRlox or IRKO cells overexpressing the insulin receptor was described previously [29]. Cells were washed three times with phosphate-buffered saline and incubated in 2 ml of HEPES binding buffer (HBB: 100 mM HEPES, pH 7.4, 118 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 8.8 mM dextrose, 1% bovine serum albumin) containing 0.01 nM 125I-insulin or 125I-IGF-I with increasing concentrations (0.1–1000 nM) of the respective unlabeled ligand for 2 h at 22 °C. Immunoblots for phospho-Akt and -MAPK were scanned on a Hewlett-Packard scanjet, and bands were quantified using ImageQuant software (Molecular Dynamics)
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