FoxO transcription factors have been implicated in lipid metabolism; however, the underlying mechanisms are not well understood. Here, in an effort to elucidate such mechanisms, we examined the phenotypic consequences of liver-specific deletion of three members of the FoxO family: FoxO1, FoxO3, and FoxO4. These liver-specific triply null mice, designated LTKO, exhibited elevated triglycerides in the liver on regular chow diet. More remarkably, LTKO mice developed severe hepatic steatosis following placement on a high fat diet. Further analyses revealed that hepatic NAD ؉ levels and Sirt1 activity were The O family members of Forkhead transcription factors (FoxOs) 3 are critical downstream effectors of the insulin/IGF-1 signaling pathway and play important roles in cellular growth and differentiation, protection against oxidative stress, and metabolic regulation (1-4). As a prototypical member of the FoxO family that includes four mammalian genes (FoxO1/3/ 4/6; FoxO2 is a pseudogene of FoxO3, and FoxO5 is the fish ortholog of FoxO3), FoxO1 has been implicated in nutrient and energy homeostasis (1-5). Although its function in glucose metabolism has been well documented (6 -12), the role of FoxO1 in hepatic lipid metabolism remains an area of active investigation (6,10,(12)(13)(14)(15)(16)(17). Adenovirus-mediated overexpression of the constitutively nuclear FoxO1 mutant (FoxO1ADA) increases triglycerides in the mouse liver but decreases triglycerides in the circulation (15). In line with this observation, deletion of FoxO1 in the liver of systemic insulin receptor (IR) knock-out mice reverses the development of hepatic steatosis (10). However, liver-specific FoxO1 knock-out mice manifest increased secretion of VLDL triglycerides in the streptozotocin-induced diabetic state (17). Moreover, the results from two transgenic lines harboring constitutively active FoxO1 alleles are contradictory in regard to its role in hepatic lipid metabolism (6,13,14). One line engineered with an S253A mutation (mouse FoxO1) leads to elevated serum and liver triglycerides (8, 13), whereas a triple substitution FOXO1TSS-A allele (T24A, S256A, and S319A in human FOXO1) exhibits lower levels of plasma triglycerides and normal levels of liver triglycerides (6). The latter finding is consistent with the observations from liver-specific IR or IR substrate (Irs1/2) knock-out mice, which have constitutively active FoxOs and lower levels of serum triglycerides due to deficiency of hepatic insulin signaling in these mice (12,18). In addition, liver-specific deletion of Akt2, the major inhibitory kinase for FoxOs, in the leptin-deficient ob/ob mice reduces fasted serum triglycerides and protects them from developing hepatic steatosis (19).In addition to phosphorylation by numerous kinases, such as Akt2, FoxOs are also subject to acetylation by several protein acetylases (20 -25). It is well known that FoxO acetylation can be reversed by NAD ϩ -dependent deacetylases (sirtuins, such as Sirt1 and Sirt2) (20 -25). Several sirtuins, including Sirt...

Neuronal migration is a fundamental process during the development of the cerebral cortex and is regulated by cytoskeletal components. Microtubule dynamics can be modulated by posttranslational modifications to tubulin subunits. Acetylation of ␣-tubulin at lysine 40 is important in regulating microtubule properties, and this process is controlled by acetyltransferase and deacetylase. MEC-17 is a newly discovered ␣-tubulin acetyltransferase that has been found to play a major role in the acetylation of ␣-tubulin in different species in vivo. However, the physiological function of MEC-17 during neural development is largely unknown. Here, we report that MEC-17 is critical for the migration of cortical neurons in the rat. MEC-17 was strongly expressed in the cerebral cortex during development. MEC-17 deficiency caused migratory defects in the cortical projection neurons and interneurons, and perturbed the transition of projection neurons from the multipolar stage to the unipolar/bipolar stage in the intermediate zone of the cortex. Furthermore, knockdown of ␣-tubulin deacetylase HDAC6 or overexpression of tubulin K40Q to mimic acetylated ␣-tubulin could reduce the migratory and morphological defects caused by MEC-17 deficiency in cortical projection neurons. Thus, MEC-17, which regulates the acetylation of ␣-tubulin, appears to control the migration and morphological transition of cortical neurons. This finding reveals the importance of MEC-17 and ␣-tubulin acetylation in cortical development.

Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. A detailed understanding of PTP functions in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific, cell-permeable small molecule agents. We present a stepwise focused library approach that transforms a weak and general nonhydrolyzable pTyr mimetic (F2Pmp, phosphonodifluoromethyl phenylalanine) into a highly potent and selective inhibitor of PTP-MEG2, an antagonist of hepatic insulin signaling. The crystal structures of the PTP-MEG2-inhibitor complexes provide direct evidence that potent and selective PTP inhibitors can be obtained by introducing molecular diversity into the F2Pmp scaffold to engage both the active site and unique nearby peripheral binding pockets. Importantly, the PTP-MEG2 inhibitor possesses highly efficacious cellular activity and is capable of augmenting insulin signaling and improving insulin sensitivity and glucose homeostasis in diet-induced obese mice. The results indicate that F2Pmp can be converted into highly potent and selective PTP inhibitory agents with excellent in vivo efficacy. Given the general nature of the approach, this strategy should be applicable to other members of the PTP superfamily.

Background Adenosine monophosphate-activated protein kinase (AMPK) is stimulated in embryos during diabetic pregnancy by maternal hyperglycaemia-induced embryo oxidative stress. Stimulation of AMPK disrupts embryo gene expression and causes neural tube defects. Metformin, which may be taken during early pregnancy, has been reported to stimulate AMPK activity. Thus, the benefits of improved glycaemic control could be offset by stimulated embryo AMPK activity. Here, we investigated whether metformin can stimulate AMPK activity in mouse embryos and can adversely affect embryo gene expression and neural tube defects. Methods Pregnant nondiabetic mice were administered metformin beginning on the first day of pregnancy. Activation of maternal and embryo AMPK [phospho-AMPK α (Thr172) relative to total AMPK], expression of Pax3, a gene required for neural tube closure, and neural tube defects were studied. Mouse embryonic stem cells were used as a cell culture model of embryonic neuroepithelium to study metformin effects on AMPK and Pax3 expression. Results Metformin had no effect on AMPK in embryos or maternal skeletal muscle but increased activated AMPK in maternal liver. Metformin did not inhibit Pax3 expression or increase neural tube defects. However, metformin increased activated AMPK and inhibited Pax3 expression by mouse embryonic stem cells. Mate1/Slc47a1 and Oct3/Slc22a, which encode metformin transporters, were expressed at barely detectable levels by embryos. Conclusions Although metformin can have effects associated with diabetic embryopathy in vitro, the lack of effects on mouse embryos in vivo may be due to lack of metformin transporters and indicates that the benefits of metformin on glycaemic control are not counteracted by stimulation of embryo AMPK activity and consequent embryopathy.

Previous studies have shown that diabetic embryopathy results from impaired expression of genes that are required for formation of embryonic structures. We have focused on Pax3, a gene that is expressed in embryonic neuroepithelium and is required for neural tube closure. Pax3 expression is inhibited in embryos of diabetic mice due to hyperglycemia-induced oxidative stress. DNA methylation silences developmentally expressed genes before differentiation. We hypothesized that hypomethylation of Pax3 upon neuroepithelial differentiation may be inhibited by hyperglycemia-induced oxidative stress. We tested this using embryos of pregnant hyperglycemic mice and mouse embryonic stem cells (ESC). Methylation of a Pax3 CpG island decreased upon neurulation of embryos and formation of neuronal precursors from ESC. In ESC, this was inhibited by oxidative stress. Use of short hairpin RNA in ESC demonstrated that DNA methyltransferase 3b (Dnmt3b) was responsible for methylation and silencing of Pax3 before differentiation and by oxidative stress. Although expression of Dnmt3b was not affected by oxidative stress, DNA methyltransferase activity was increased. These results indicate that hyperglycemia-induced oxidative stress stimulates Dnmt3b activity, thereby inhibiting chromatin modifications necessary for induction of Pax3 expression during neurulation and thus providing a molecular mechanism for defects caused by Pax3 insufficiency in diabetic pregnancy.