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Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKK␤, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-␣, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-␣ signaling with neutralizing antibodies to TNF-␣ or its receptors or with a dominant negative TNF-␣ peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-␣ signaling, whereas the FFA-induced increase in TNF-␣ secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-␣-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-␣ is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.

The insulin-like growth factor 1 (IGF1) receptor is closely related to the insulin receptor. However, the unique biological functions of IGF1 receptor make it a target for therapeutic intervention in human cancer. Using its isolated tyrosine kinase domain, we show that the IGF1 receptor is regulated by intermolecular autophosphorylation at three sites within the kinase activation loop. Steady-state kinetic analyses of the isolated phosphorylated forms of the IGF1 receptor kinase (IGF1RK) reveal that each autophosphorylation event increases enzyme turnover number and decreases Km for ATP and peptide. We have determined the 2.1 A-resolution crystal structure of the tris-phosphorylated form of IGF1RK in complex with an ATP analog and a specific peptide substrate. The structure of IGF1RK reveals how the enzyme recognizes peptides containing hydrophobic residues at the P+1 and P+3 positions and how autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Although the nucleotide binding cleft is conserved between IGF1RK and the insulin receptor kinase, sequence differences in the nearby interlobe linker could potentially be exploited for anticancer drug design.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays a critical role in regulating insulin sensitivity and glucose homeostasis. In this study, we identified highly efficient small interfering RNA (siRNA) sequences and used lentiviral short hairpin RNA and electroporation of siRNAs to deplete PPAR-γ from 3T3-L1 adipocytes to elucidate its role in adipogenesis and insulin signaling. We show that PPAR-γ knockdown prevented adipocyte differentiation but was not required for maintenance of the adipocyte differentiation state after the cells had undergone adipogenesis. We further demonstrate that PPAR-γ suppression reduced insulin-stimulated glucose uptake without affecting the early insulin signaling steps in the adipocytes. Using dual siRNA strategies, we show that this effect of PPAR-γ deletion was mediated by both GLUT4 and GLUT1. Interestingly, PPAR-γ-depleted cells displayed enhanced inflammatory responses to TNF-α stimulation, consistent with a chronic anti-inflammatory effect of endogenous PPAR-γ. In summary, 1) PPAR-γ is essential for the process of adipocyte differentiation but is less necessary for maintenance of the differentiated state, 2) PPAR-γ supports normal insulin-stimulated glucose transport, and 3) endogenous PPAR-γ may play a role in suppression of the inflammatory pathway in 3T3-L1 cells.

MBX-102/JNJ39659100 (MBX-102) is in clinical development as an oral glucose-lowering agent for the treatment of type 2 diabetes. MBX-102 is a nonthiazolidinedione (TZD) selective partial agonist of peroxisome proliferator-activated receptor (PPAR)-gamma that is differentiated from the TZDs structurally, mechanistically, preclinically and clinically. In diabetic rodent models, MBX-102 has insulin-sensitizing and glucose-lowering properties comparable to TZDs without dose-dependent increases in body weight. In vitro, in contrast with full PPAR-gamma agonist treatment, MBX-102 fails to drive human and murine adipocyte differentiation and selectively modulates the expression of a subset of PPAR-gamma target genes in mature adipocytes. Moreover, MBX-102 does not inhibit osteoblastogenesis of murine mesenchymal cells. Compared with full PPAR-gamma agonists, MBX-102 displays differential interactions with the PPAR-gamma ligand binding domain and possesses reduced ability to recruit coactivators. Interestingly, in primary mouse macrophages, MBX-102 displays enhanced antiinflammatory properties compared with other PPAR-gamma or alpha/gamma agonists, suggesting that MBX-102 has more potent transrepression activity. In summary, MBX-102 is a selective PPAR-gamma modulator with weak transactivation but robust transrepression activity. MBX-102 exhibits full therapeutic activity without the classical PPAR-gamma side effects and may represent the next generation insulin sensitizer.

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MBX-102/JNJ39659100 (MBX-102) is in clinical development as an oral glucose lowering agent for the treatment of type 2 diabetes. MBX-102 is a non-thiazolidinedione (TZD) selective partial agonist of PPAR-γ that is differentiated from the TZDs structurally, mechanistically, pre-clinically and clinically. In diabetic rodent models, MBX-102 has insulin sensitizing and glucose lowering properties comparable to TZDs without dose-dependent increases in body weight. In vitro, in contrast with full PPAR-γ agonist treatment, MBX-102 fails to drive human and murine adipocyte differentiation and selectively modulates the expression of a subset of PPAR-γ target genes in mature adipocytes. Moreover, MBX-102 does not inhibit oteoblastogenesis of murine mesenchymal cells. Compared to full PPAR-γ agonists, MBX-102 displays differential interactions with the PPAR-γ ligand binding domain (LBD) and possesses reduced ability to recruit coactivators. Interestingly, in primary mouse macrophages, MBX-102 displays enhanced anti-inflammatory properties compared to other PPAR-γ or α/γ agonists suggesting that MBX-102 has more potent transrepression activity. In summary, MBX-102 is a selective PPAR-γ modulator with weak transactivation but robust transrepression activity. MBX-102 exhibits full therapeutic activity without the classical PPAR-γ side effects and may represent the next generation insulin sensitizer.

No abstract