Insulin resistance is a hallmark of type 2 diabetes, and many insights into the functions of insulin have been gained through the study of mice lacking the IR. To gain a better understanding of the role of insulin action in the brain versus peripheral tissues, we created 2 mouse models with inducible IR inactivation, 1 in all tissues including brain (IR Δwb ), and 1 restricted to peripheral tissues (IR Δper ). While downregulation of IR expression resulted in severe hyperinsulinemia in both models, hyperglycemia was more pronounced in IR Δwb mice. Both strains displayed a dramatic upregulation of hepatic leptin receptor expression, while only IR Δper mice displayed increased hepatic Stat3 phosphorylation and Il6 expression. Despite a similar reduction in IR expression in white adipose tissue (WAT) mass in both models, IR Δwb mice had a more pronounced reduction in WAT mass and severe hypoleptinemia. Leptin replacement restored hepatic Stat3 phosphorylation and normalized glucose metabolism in these mice, indicating that alterations in glucose metabolism occur largely as a consequence of lipoathrophy upon body-wide IR deletion. Moreover, chronic intracerebroventricular insulin treatment of control mice increased fat mass, fat cell size, and adipose tissue lipoprotein lipase expression, indicating that CNS insulin action promotes lipogenesis. These studies demonstrate that central insulin action plays an important role in regulating WAT mass and glucose metabolism via hepatic Stat3 activation.

PLRG1, an evolutionarily conserved component of the spliceosome, forms a complex with Pso4/SNEV/Prp19 and the cell division and cycle 5 homolog (CDC5L) that is involved in both pre-mRNA splicing and DNA repair. Here, we show that the inactivation of PLRG1 in mice results in embryonic lethality at 1.5 days postfertilization. Studies of heart-and neuron-specific PLRG1 knockout mice further reveal an essential role of PLRG1 in adult tissue homeostasis and the suppression of apoptosis. PLRG1-deficient mouse embryonic fibroblasts (MEFs) fail to progress through S phase upon serum stimulation and exhibit increased rates of apoptosis. PLRG1 deficiency causes enhanced p53 phosphorylation and stabilization in the presence of increased ␥-H2AX immunoreactivity as an indicator of an activated DNA damage response. p53 downregulation rescues lethality in both PLRG1-deficient MEFs and zebrafish in vivo, showing that apoptosis resulting from PLRG1 deficiency is p53 dependent. Moreover, the deletion of PLRG1 results in the relocation of its interaction partner CDC5L from the nucleus to the cytoplasm without general alterations in pre-mRNA splicing. Taken together, the results of this study identify PLRG1 as a critical nuclear regulator of p53-dependent cell cycle progression and apoptosis during both embryonic development and adult tissue homeostasis.Mammalian pleiotropic regulator (PLRG1) belongs to a highly conserved family of seven WD40 domain-containing proteins in eukaryotes (2, 4). The founding members of this protein family, PRL1 and PRL2, were identified first by T-DNA tagging in Arabidopsis thaliana, where PRL1 deficiency augments the sensitivity to growth hormones, stimulates the accumulation of sugars and starch in leaves, and inhibits root elongation (37). More recently, it has been demonstrated that the A. thaliana CDC5/PRL1 complex is essential for plant innate immunity (39).Human PLRG1 initially was identified as a subunit of spliceosomal complexes purified from HeLa nuclear extracts by coimmunoprecipitation with CDC5L (2). Indeed, interaction between PLRG1 and CDC5L appears essential for pre-mRNA processing, as peptides inhibiting CDC5L-PLRG1 complex formation efficiently block pre-mRNA splicing (3).The CDC5L-PLRG1 complex also was found to interact with the WRN protein, which is deficient in Werner syndrome, a rare autosomal recessive human disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancer (1,18,28,34,53). The WRN protein has exonuclease and RecQ helicase activity, interacts with multiple proteins of the DNA replication complex, and is crucial for DNA synthesis and DNA repair (12,30,31,41,43,53). Thus, PLRG1 offers an interesting link between the control of pre-mRNA splicing and DNA metabolism.To elucidate the in vivo role of PLRG1 in vertebrates, we have generated conventional and conditional knockout (KO) mice for PLRG1, as well as antisense-mediated plrg1 knockdown zebrafish embryos and cell lines with an inducible inactivation of PLRG1...

Most B lymphocytes in normal individuals express two classes of cell-surface immunoglobulins, IgM and IgD. The specificity of the two antigen receptors is identical since they are produced by transcription and differential splicing of the same variable region gene segment to the heavy-chain constant region gene segments for both ,u and 8 heavy chains. B lymphocytes expressing other immunoglobulin isotypes, IgG, IgA, or IgE, are rare and not well characterized. Particularly controversial is the molecular mechanism of their isotype switch. Here we use high-gradient magnetic cell sorting and fluorescence-activated cell sorting to purify surface IgAlbearing B lymphocytes from human blood for cellular and molecular analysis. These cells express no immunoglobulin class other than IgAl and are a relatively uniform population with regard to expression of other cell-surface molecules. They are resting cells in terms of cell cycle and activation marker analysis. The molecular basis for class switching in the IgAl1 cells is not differential transcription or splicing. Rather, switch recombination involving deletion of DNA has occurred on both immunoglobulin heavy-chain gene loci, including the allefically excluded one, and appears to have been directed to IgAl under normal physiological conditions.

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