Transcription factor Neurod1 is required for enteroendocrine progenitor differentiation and maturation. Several earlier studies indicated that ectopic expression of Neurod1 converted non- neuronal cells into neurons. However, the functional consequence of ectopic Neurod1 expression has not been examined in the GI tract, and it is not known whether Neurod1 can similarly switch cell fates in the intestine. We generated a mouse line that would enable us to conditionally express Neurod1 in intestinal epithelial cells at different stages of differentiation. Forced expression of Neurod1 throughout intestinal epithelium increased the number of EECs as well as the expression of EE specific transcription factors and hormones. Furthermore, we observed a substantial reduction of Paneth cell marker expression, although the expressions of enterocyte-, tuft- and goblet-cell specific markers are largely not affected. Our earlier study indicated that Neurog3+ progenitor cells give rise to not only EECs but also Goblet and Paneth cells. Here we show that the conditional expression of Neurod1 restricts Neurog3+ progenitors to adopt Paneth cell fate, and promotes more pronounced EE cell differentiation, while such effects are not seen in more differentiated Neurod1+ cells. Together, our data suggest that forced expression of Neurod1 programs intestinal epithelial cells more towards an EE cell fate at the expense of the Paneth cell lineage and the effect ceases as cells mature to EE cells.

Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15 (-/-) mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15 (-/-) liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1alpha expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action.

RATIONALE: Mitochondria, although required for cellular ATP production, are also known to have other important functions that may include modulating cellular responses to environmental stimuli. However, the mechanisms whereby mitochondria impact cellular phenotype are not yet clear. OBJECTIVE: To determine how mitochondria impact endothelial cell function. METHODS AND RESULTS: We report here that stimuli for endothelial cell proliferation evoke strong upregulation of mitochondrial uncoupling protein 2 (UCP2). Analysis in silico indicated increased UCP2 expression is common in highly proliferative cell types, including cancer cells. Upregulation of UCP2 was critical for controlling mitochondrial membrane potential (Deltapsi) and superoxide production. In the absence of UCP2, endothelial growth stimulation provoked mitochondrial network fragmentation and premature senescence via a mechanism involving superoxide-mediated p53 activation. Mitochondrial network fragmentation was both necessary and sufficient for the impact of UCP2 on endothelial cell phenotype. CONCLUSIONS: These data identify a novel mechanism whereby mitochondria preserve normal network integrity and impact cell phenotype via dynamic regulation of UCP2.

Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.

Pathogenic members of the Yersinia genus require the translocator protein LcrV for proper function of the type III secretion apparatus, which is crucial for virulence. LcrV has also been reported to play an independent immunosuppressive role via the induction of interleukin-10 (IL-10) through stimulation of Toll-like receptor 2 (TLR2). To investigate the LcrV-TLR2 interaction in vitro, His-tagged recombinant LcrV (rLcrV) from Yersinia pestis was cloned and expressed in Escherichia coli and purified through Ni-nitrilotriacetic acid column chromatography. High concentrations (5 microg/ml) of rLcrV stimulated TLR2 in vitro. Fractionation of rLcrV preparations via gel filtration revealed that only a minor component consisting of high-molecular-weight multimers or aggregates has TLR2 stimulating activity. Dimer and tetramer forms of rLcrV, which constitute the bulk of the material, do not have this activity. To investigate the potential role of LcrV/TLR2 in plague pathogenesis, we infected wild-type and TLR2(-/-) mice with virulent Y. pestis. No discernible difference between the two mouse strains in severity of disease or kinetics of survival after subcutaneous challenge was observed. IL-6, tumor necrosis factor, and IL-10 levels from spleen homogenates; bacterial load; and the extent of inflammation observed in organs from mice infected intravenously were also indistinguishable in both mouse strains. Taken together, our data indicate that the most abundant molecular species of Y. pestis LcrV do not efficiently activate TLR2-signaling and that TLR2-mediated immunomodulation is unlikely to play a significant role in plague.