FOXO1 and peroxisome proliferator-activated receptor-␥ (PPAR␥) are crucial transcription factors that regulate glucose metabolism and insulin responsiveness in insulin target tissues. We have shown that, in primary rat adipocytes, both factors regulate transcription of the insulin-responsive GLUT4 gene and that PPAR␥2 detachment from the GLUT4 promoter upon thiazolidinedione binding up-regulates GLUT4 gene expression, thus increasing insulin sensitivity (Armoni, M., Kritz, N., Harel, C., Bar-Yoseph, F., Chen, H., Quon, M. J., and Karnieli, E. (2003) J. Biol. Chem. 278, 30614 -30623). However, the mechanisms regulating PPAR␥ gene transcription are largely unknown. We studied the effects of FOXO1 on human PPAR␥ gene expression in primary rat adipocytes and found that both genes are endogenously expressed. FOXO1 coexpression dosedependently repressed transcription from either the PPAR␥1 or PPAR␥2 promoter reporter by 65%, whereas insulin (100 nM, 20 -24 h) either partially or completely reversed this effect. Phosphorylation-defective FOXO1 mutants T24A, S256A, S319A, and T24A/S256A/S319A still repressed the PPAR␥1 promoter and partially lost their effects on the PPAR␥2 promoter in either basal or insulin-stimulated cells. Use of DNA binding-defective FOXO1 (H215R) indicated that this domain is crucial for FOXO1 repression of the PPAR␥2 (but not PPAR␥1) promoter. Progressive 5-deletion and gel retardation analyses revealed that this repression involves direct and specific binding of FOXO1 to the PPAR␥2 promoter; chromatin immunoprecipitation analysis confirmed that this binding occurs in cellulo. We suggest a novel paradigm to increase insulin sensitivity in adipocytes in which FOXO1 repression of PPAR␥, the latter being a repressor of the GLUT4 promoter, consequently leads to GLUT4 derepression/up-regulation, thus enhancing cellular insulin sensitivity. The newly identified FOXO1-binding site on the PPAR␥2 promoter may serve as a therapeutic target for type 2 diabetes.The peroxisome proliferator-activated receptor (PPAR) 2 family of nuclear receptors and the FOXO (forkhead box class O) family of winged helix/forkhead box factors are two key families of transcription factors that dominate the regulation of glucose metabolism and insulin responsiveness in insulin target tissues. Members of both families are crucial for a multitude of biological processes, including the cell cycle, cell death, differentiation, and metabolism, and have prominent roles in insulin signaling pathways. A convergence of nuclear receptors and forkhead pathways in general and of FOXO1 and PPAR␥ in particular has been implicated in the pathophysiological states of insulin resistance and diabetes, supporting the importance of these transcription factors (1, 2). However, despite their importance to glucose homeostasis and adipocyte differentiation, the molecular mechanism(s) regulating transcription of the PPAR␥ gene and the roles of both PPAR␥ and FOXO1 transcription factors in these processes are not fully known.The PPAR family of ligand-activ...

Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wildtype mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-␣-treated cells. In NIH-3T3 IR cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-␣. Using mass spectrometry, we identified Ser 24 in the pleckstrin homology (PH) domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser 24 ) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-␣ or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser 24 , we found that interleukin-1 or TNF-␣ treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser 24 . We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-␣-regulated phosphorylation at Ser 24 in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance.Biochemical, physiological, and epidemiological studies implicate pro-inflammatory cytokines (e.g. TNF-␣, 1 IL-1␤, and IL-6) in the development of insulin resistance and the pathophysiology of type 2 diabetes and obesity (1-8). These studies suggest an intriguing link between inflammation and metabolic dysregulation. Indeed, IB kinase ␤ (IKK␤), a critical mediator of inflammatory signaling pathways activating NF-B, has been identified as an important inhibitor of metabolic insulin signaling pathways (9 -12). Inactivation of IKK␤ signaling increases insulin sensitivity, whereas overexpression of IKK␤ or activation of IKK␤ by pro-inflammatory cytokines (e.g. TNF-␣) leads to insulin resistance (9, 12). Similarly, JNK is another inflammatory signaling molecule that may play a role in the insulin resistance of obesity (13). One potential explanation for these observations is cross-talk between inflammatory signaling and metabolic insulin signaling pathways.Metabolic actions of insulin such as enhanced glucose uptake into skeletal muscle and adipose tissue are regulated by activation of the insulin receptor tyrosine kinase and subsequent tyrosine phosphorylation o...