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Preadipocyte factor-1 (Pref-1) is made as a transmembrane protein containing EGF-repeats at the extracellular domain that can be cleaved to generate a biologically active soluble form. Pref-1 is found in islet β-cells and its level has been reported to increase in neonatal rat islets upon growth hormone treatment. We found here that Pref-1 can promote growth of pancreatic tumor derived AR42J cells. To examine Pref-1 function in pancreatic islets invivo, we generated transgenic mouse lines overexpressing the Pref-1/hFc in islet β-cells using rat insulin II promoter (RIP). These transgenic mice exhibit an increase in islet mass with higher proportion of larger islets in pancreas compared to wild-type littermates. This is in contrast to pancreas from Pref-1 null mice that show higher proportion of smaller islets. Insulin expression and insulin secretion from pancreatic islets from RIP-Pref-1/hFc transgenic mice are increased also. Thus, RIP-Pref-1/hFc transgenic mice show normal glucose levels but with higher plasma insulin levels in both fasting and fed conditions. These mice show improved glucose tolerance. Taken together, we conclude Pref-1 as a positive regulator of islet β-cells and insulin production.

SummaryPref-1 is an EGF-repeat-containing protein that inhibits adipocyte differentiation. To better understand the origin and development of white adipose tissue (WAT), we generated transgenic mouse models for transient or permanent fluorescent labeling of cells using the Pref-1 promoter, facilitating inducible ablation. We show that Pref-1-marked cells retain proliferative capacity and are very early adipose precursors, prior to expression of Zfp423 or PPARγ. In addition, the Pref-1-marked cells establish that adipose precursors are mesenchymal, but not endothelial or pericytal, in origin. During embryogenesis, Pref-1-marked cells first appear in the dorsal mesenteric region as early as embryonic day 10.5 (E10.5). These cells become lipid-laden adipocytes at E17.5 in the subcutaneous region, whereas visceral WAT develops after birth. Finally, ablation of Pref-1-marked cells prevents not only embryonic WAT development but also later adult adipose expansion upon high-fat feeding, demonstrating the requirement of Pref-1 cells for adipogenesis.

A main function of white adipose tissue is to release fatty acids from stored triacylglycerol for other tissues to use as an energy source. Whereas endocrine regulation of lipolysis has been extensively studied, autocrine and paracrine regulation is not well understood. Here we describe the role of the newly identified major adipocyte phospholipase A(2), AdPLA (encoded by Pla2g16, also called HREV107), in the regulation of lipolysis and adiposity. AdPLA-null mice have a markedly higher rate of lipolysis owing to increased cyclic AMP levels arising from the marked reduction in the amount of adipose prostaglandin E(2) that binds the Galpha(i)-coupled receptor, EP3. AdPLA-null mice have markedly reduced adipose tissue mass and triglyceride content but normal adipogenesis. They also have higher energy expenditure with increased fatty acid oxidation within adipocytes. AdPLA-deficient ob/ob mice remain hyperphagic but lean, with increased energy expenditure, yet have ectopic triglyceride storage and insulin resistance. AdPLA is a major regulator of adipocyte lipolysis and is crucial for the development of obesity.

Vance et al. provide genetic proof for the specificity and essentiality of NAIP proteins for inflammasome responses to specific bacterial ligands in vivo., NLRs (nucleotide-binding domain [NBD] leucine-rich repeat [LRR]–containing proteins) exhibit diverse functions in innate and adaptive immunity. NAIPs (NLR family, apoptosis inhibitory proteins) are NLRs that appear to function as cytosolic immunoreceptors for specific bacterial proteins, including flagellin and the inner rod and needle proteins of bacterial type III secretion systems (T3SSs). Despite strong biochemical evidence implicating NAIPs in specific detection of bacterial ligands, genetic evidence has been lacking. Here we report the use of CRISPR/Cas9 to generate Naip1−/− and Naip2−/− mice, as well as Naip1-6Δ/Δ mice lacking all functional Naip genes. By challenging Naip1−/− or Naip2−/− mice with specific bacterial ligands in vivo, we demonstrate that Naip1 is uniquely required to detect T3SS needle protein and Naip2 is uniquely required to detect T3SS inner rod protein, but neither Naip1 nor Naip2 is required for detection of flagellin. Previously generated Naip5−/− mice retain some residual responsiveness to flagellin in vivo, whereas Naip1-6Δ/Δ mice fail to respond to cytosolic flagellin, consistent with previous biochemical data implicating NAIP6 in flagellin detection. Our results provide genetic evidence that specific NAIP proteins function to detect specific bacterial proteins in vivo.

OBJECTIVE To investigate the role of desnutrin in adipose tissue triacylglycerol (TAG) and fatty acid metabolism. RESEARCH DESIGN AND METHODS We generated transgenic mice overexpressing desnutrin (also called adipose triglyceride lipase [ATGL]) in adipocytes (aP2-desnutrin) and also performed adenoviral-mediated overexpression of desnutrin in 3T3-L1CARΔ1 adipocytes. RESULTS aP2-desnutrin mice were leaner with decreased adipose tissue TAG content and smaller adipocyte size. Overexpression of desnutrin increased lipolysis but did not result in increased serum nonesterified fatty acid levels or ectopic TAG storage. We found increased cycling between diacylglycerol (DAG) and TAG and increased fatty acid oxidation in adipocytes from these mice, as well as improved insulin sensitivity. CONCLUSIONS We show that by increasing lipolysis, desnutrin overexpression causes reduced adipocyte TAG content and attenuation of diet-induced obesity. Desnutrin-mediated lipolysis promotes fatty acid oxidation and re-esterification within adipocytes.

The production of distinct sets of T cell receptor (TCR) gammadelta(+) T cells occurs in an ordered fashion in thymic development. The Vgamma3 and Vgamma4 genes, located downstream in the TCRgamma Cgamma1 gene cluster, are expressed by the earliest waves of developing TCRgammadelta(+) T cells in the fetal thymus, destined for intraepithelial locations. Upstream Vgamma2 and Vgamma5 genes are expressed in later waves in the adult and constitute most TCRgammadelta(+) T cells in secondary lymphoid tissue. This developmental pattern is caused in part by a preference for rearrangements of the downstream Vgamma3 and Vgamma4 genes in the early fetal stage, which switches to a preference for rearrangements of the upstream Vgamma2 and Vgamma5 gene rearrangements in the adult. Our gene targeting studies show that the downstream Vgamma genes rearrange preferentially in the early fetal thymus because of their downstream location, independent of promoter or recombination signal sequences and unrelated to the extent of germline transcription. Remarkably, gene deletion studies show that the downstream Vgamma genes competitively inhibit upstream Vgamma rearrangements at the fetal stage. These data provide a mechanism for specialization of the fetal thymus for the production of T cells expressing specific Vgamma genes.

Uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis and, upon cold exposure, is induced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a transcriptional activator of UCP1 as wellas PGC1α, thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature and energy expenditure and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics.

Although Notch plays a crucial role in T cell development, regulation of Notch signaling in the thymus is not well understood. Kuzbanian, an ADAM protease, has been implicated in the cleavage of both Notch receptors and the Notch ligand, Delta. In this study we show that the expression of a dominant-negative form of Kuzbanian (dnKuz) leads to reduced TCRβ expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. Our data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components.

BAF and PBAF are two related mammalian chromatin remodeling complexes essential for gene expression and development. PBAF, but not BAF, is able to potentiate transcriptional activation in vitro mediated by nuclear receptors, such as RXRalpha, VDR, and PPARgamma. Here we show that the ablation of PBAF-specific subunit BAF180 in mouse embryos results in severe hypoplastic ventricle development and trophoblast placental defects, similar to those found in mice lacking RXRalpha and PPARgamma. Embryonic aggregation analyses reveal that in contrast to PPARgamma-deficient mice, the heart defects are likely a direct result of BAF180 ablation, rather than an indirect consequence of trophoblast placental defects. We identified potential target genes for BAF180 in heart development, such as S100A13 as well as retinoic acid (RA)-induced targets RARbeta2 and CRABPII. Importantly, BAF180 is recruited to the promoter of these target genes and BAF180 deficiency affects the RA response for CRABPII and RARbeta2. These studies reveal unique functions of PBAF in cardiac chamber maturation.

Adipocyte-specific secretory factor (ADSF)/resistin is a small cysteine-rich protein secreted from adipose tissue that belongs to a gene family found in inflammatory zone (FIZZ) or found in resistin-like molecule (RELM). ADSF has been implicated in modulating adipogenesis and insulin resistance. To examine the long-term function of ADSF in adipogenesis and glucose homeostasis, we constructed an expression vector for a dominant inhibitory form of ADSF by fusing it to the human IgGγ constant region (hFc). ADSF-hFc not only homodimerizes but heterooligomerizes with ADSF/resistin and prevents ADSF/resistin inhibition of adipocyte differentiation of 3T3-L1 cells in a dominant negative manner. Transgenic mice overexpressing ADSF-hFc in adipose tissue show increased adiposity with elevated expression of adipocyte markers as well as enlarged adipocyte size. This finding clearly demonstrates in vivo the inhibitory role of ADSF in adipogenesis. ADSF-hFc transgenic mice with impaired ADSF function exhibit improved glucose tolerance and insulin sensitivity either on chow or high-fat diets. Because of the enhanced adipocyte differentiation, the ADSF-hFc transgenic mice show increased expression of leptin and adiponectin in adipose tissue. The elevated circulating levels for these adipocyte-derived hormones with decreased plasma triglyceride and free fatty acid levels may account for the improved glucose and insulin tolerance in these transgenic mice.

Mice deficient for the B cell–restricted transcription factor Pax5 show a defect in the VH to DJH rearrangement step of immunoglobulin heavy chain gene assembly even though the expression of the V(D)J recombinase is not diminished in Pax5−/− pro–B cells. To investigate whether Pax5 is limiting for VH to DJH rearrangement, we generated transgenic mice which express Pax5 in developing thymocytes. We show that enforced expression of Pax5 in thymocytes results in a partial block in T cell development due to defective pre-TCR signaling in β-selection. Moreover, our results demonstrate that expression of Pax5 in early thymocytes is sufficient to induce VH to DJH rearrangements in CD4+CD8+ T cells and lead us to suggest that Pax5 may play a direct role in the lineage-specific regulation of immunoglobulin heavy chain gene rearrangement.

Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell–specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre–T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.

Developmentally regulated V(D)J recombination profoundly influences immune repertoires, but the underlying mechanisms are poorly understood. In the endogenous T cell receptor Cgamma1 cluster, the 3' Vgamma3 gene (closest to Jgamma1) rearranges preferentially in the fetal period whereas rearrangement of the 5' Vgamma2 gene predominates in the adult. Reversing the positions of the Vgamma2 and Vgamma3 genes in a genomic transgene resulted in decreased rearrangement of the now 5' Vgamma3 gene in the fetal thymus and increased rearrangement of the now 3' Vgamma2 gene. The reversed rearrangement pattern was not accompanied by significant changes in chromatin accessibility of the relocated Vgamma genes. The results support a model in which the 3' location is the key determinant of rearrangement in the fetus, after which there is a promoter-dependent inactivation of Vgamma3 rearrangement in favor of Vgamma2 rearrangement.

12 pages, 7 figures., Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5' deletions as well as site-specific mutations of the FAS promoter. We show that the -332 and -65 E-boxes are bound by USF in both fasted and refed mice, while the -150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the -150 SRE or the -65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the -150 SRE but, unexpectedly, of the -65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the -150 SRE and that USF binding to the -65 E-box is also required for SREBP binding and activation of the FAS promoter., The work was supported by NIH grant DK36264 to H.S.S.

FADD is an adaptor known to transmit apoptotic signals from members of the tumor necrosis factor receptor family. We show here that FADD has a domain implicated in cell proliferation. Mice bearing the Asp mutation in the serine 191 phosphorylation site are runted and anemic and display splenomegaly. Apoptosis is unimpaired in these mice, but they exhibit many immune developmental problems indicative of proliferative defects. Mutant FADD T cells are defective in cell cycle progression, suggesting that regulation of phosphorylation at serine 191 is essential for growth/proliferation. Remarkably, serine 191 is conserved among mammalian FADD proteins, but this C-terminal region is absent in lower organisms, suggesting that FADD acquired a domain during evolution, rendering it a “proliferation-apoptosis coupler” that balances cell proliferation and apoptosis.

Many mammalian genes, including those encoding antigen receptors, contain more than one enhancer element. Deleting one element often does not prevent expression, but functional redundancy has never been directly demonstrated by gene targeting of multiple elements. We demonstrate that simultaneous deletion of two enhancer/LCR-like elements in the TCR Cgamma1 cluster, HsA and 3'E(Cgamma1), severely diminishes TCRgamma transcription, selectively impairs development of gammadelta thymocyte subsets, but only modestly reduces TCRgamma gene rearrangement, while deletion of each element separately has little effect. In contrast to these results in thymocytes, deletion of HsA alone reduces transcription of one Vgamma gene specifically in peripheral gammadelta T cells. Thus, the two elements exhibit functional redundancy in thymocytes but also have unique functions in other settings.

Cancer develops by a multistep process during which cells acquire characteristics that allow them to evade apoptosis and proliferate unchecked. Sequential acquisition of genetic alterations drives this process but also causes cellular stress, frequently prompting cells to enter a premalignant period during which they mount a defense against transformation. T cell-specific deletion of the tumor suppressor PTEN in mice induces premalignancy in the thymus and development of CD4(+) T-cell lymphomas in the periphery. Here we sought to identify factors mediating the cellular defense against transformation during the premalignant period. We identified several microRNAs upregulated specifically in premalignant thymocytes, including miR-146a, miR-146b, and the miR-183/96/182 cluster. CD4-driven T cell-specific transgenic overexpression of mir-146a and mir-146b significantly delayed PTEN-deficient lymphomagenesis and delayed c-myc oncogene induction, a key driver of transformation in PTEN-deficient T-cell malignancies. We found that miR-146a and miR-146b targeting of Traf6 attenuates TCR signaling in the thymus and inhibits downstream NF-κB-dependent induction of c-myc. Additionally, c-myc repression in mature CD4 T cells by miR-146b impaired TCR-mediated proliferation. Hence, we have identified 2 miRNAs that are upregulated as part of the cellular response against transformation that, when overrepresented, can effectively inhibit progression to malignancy in the context of PTEN deficiency.

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.

We have successfully used antisense RNA to inhibit replication of the mouse hepatitis virus (MHV) in a cell culture system. MHV is a single-stranded RNA virus of positive polarity. Mouse L2 cells were stably transfected with an antisense construct that targets regions of genes 5 and 6 of the virus. High levels of expression from this construct, which is under control of the human elongation factor 1 alpha promoter, were found. After infection of the antisense cell lines with MHV, replication of the virus was significantly reduced compared with control cells. In a viral plaque assay, smaller plaques were found in the antisense cell lines. In addition, up to a 92% inhibition in the number of viral particles produced in one antisense cell line could be seen. This inhibitory effect decreased at longer (16 hour) infection times. It was possible to both increase the amount of inhibition and prolong the inhibitory effect by reducing the multiplicity of infection. Our results suggest that antisense RNA may be an effective tool to slow down progression of MHV infection in mice.

Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4(+)CD8(+) thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4(+)CD8(+) thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4(+)CD8(+) thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4(+)CD8(+) thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis.

The PI3K-AKT pathway can mediate diverse biological responses and is crucial for optimal immune responses and lymphocyte development. Deletion of PI3K subunits or AKT leads to blockage of T-cell development at the TCR-beta checkpoint. Studies with over-expression of constitutively activated AKT have implicated this pathway in anti-apoptosis of developing thymocytes and in development of regulatory T cells. However, the role of endogenous PI3K-AKT in T-cell development beyond the TCR-beta checkpoint remains unclear. Here, we inhibited the endogenous PI3K-AKT pathway in thymocytes after double negative stages by expressing the negative regulator, PTEN. These mice exhibit normal early T-cell development, but the transition from intermediate single positive to double positive (DP) thymocytes is inhibited, leading to a significantly decreased number of DP, single positive thymocytes and peripheral T cells. Proliferation of peripheral T cells is reduced but apoptosis of DP cells and subsequent T-cell maturation, including regulatory T cells, are normal. AKT phosphorylation can be readily observed in most WT T-cell compartments but not DP thymocytes in response to TCR activation. Thus, the PI3K-AKT pathway is crucial for the transition of intermediate single positive to DP thymocytes but is dispensable for apoptosis and maturation of developing thymocytes.

Substantial evidence exists that many tumors can be specifically recognized by CD8 + T lymphocytes. The definition of antigens targeted by these cells is paramount for the development of effective immunotherapeutic strategies for treating human cancers. In a screen for endogenous tumor-associated T cell responses in a primary mouse model of prostatic adenocarcinoma, we identified a naturally arising CD8 + T cell response that is reactive to a peptide derived from histone H4. Despite the ubiquitous nature of histones, T cell recognition of histone H4 peptide was specifically associated with the presence of prostate cancer in these mice. Thus, the repertoire of antigens recognized by tumor-infiltrating T cells is broader than previously thought and includes peptides derived from ubiquitous self antigens that are normally sequestered from immune detection.

Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection. Expression of Nur77 in thymocytes and cell lines leads to apoptosis through a mechanism that remains unclear. In some cell lines, Nur77 was reported to act through a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release. However, we show here that Nur77-mediated apoptosis in thymocytes does not involve cytoplasmic cytochrome c release and cannot be rescued by Bcl-2. Microarray analysis shows that Nur77 induces many genes, including two novel genes (NDG1, NDG2) and known apoptotic genes FasL and TRAIL. Characterization of NDG1 and NDG2 indicates that NDG1 initiates a novel apoptotic pathway in a Bcl-2-independent manner. Thus Nur77-mediated apoptosis in T cells involves Bcl-2 independent transcriptional activation of several known and novel apoptotic pathways.

Major histocompatibility complex (MHC) class I molecules display tens of thousands of peptides on the cell surface, derived from virtually all endogenous proteins, for inspection by cytotoxic T cells (CTLs). We show that, in normal mouse cells, MHC I molecules present a peptide encoded in the 3′ “untranslated” region. Despite its rarity, the peptide elicits CTL responses and induces self-tolerance, establishing that immune surveillance extends well beyond conventional polypeptides. Furthermore, translation of this cryptic peptide occurs by a previously unknown mechanism that decodes the CUG initiation codon as leucine rather than the canonical methionine.

Preadipocyte factor-1 (Pref-1) is a transmembrane protein highly expressed in preadipocytes. Pref-1 expression is, however, completely abolished in adipocytes. The extracellular domain of Pref-1 undergoes two proteolytic cleavage events that generate 50 and 25 kDa soluble products. To understand the function of Pref-1, we generated transgenic mice that express the full ectodomain corresponding to the large cleavage product of Pref-1 fused to human immunoglobulin-gamma constant region. Mice expressing the Pref-1/hFc transgene in adipose tissue, driven by the adipocyte fatty acid-binding protein (aP2, also known as aFABP) promoter, showed a substantial decrease in total fat pad weight. Moreover, adipose tissue from transgenic mice showed reduced expression of adipocyte markers and adipocyte-secreted factors, including leptin and adiponectin, whereas the preadipocyte marker Pref-1 was increased. Pref-1 transgenic mice with a substantial, but not complete, loss of adipose tissue exhibited hypertriglyceridemia, impaired glucose tolerance, and decreased insulin sensitivity. Mice expressing the Pref-1/hFc transgene exclusively in liver under the control of the albumin promoter also showed a decrease in adipose mass and adipocyte marker expression, suggesting an endocrine mode of action of Pref-1. These findings demonstrate the inhibition of adipogenesis by Pref-1 in vivo and the resulting impairment of adipocyte function that leads to the development of metabolic abnormalities.

FADD is an adapter protein that was originally isolated as a transducer of apoptotic signals for death domain-containing receptors. However, FADD-deficient mice are embryonic lethal and FADD(-/-) T cells developed from FADD(-/-) embryonic stem cells in the RAG-1(-/-) hosts lack the full potential to proliferate when stimulated through their T-cell receptor complex, suggesting that FADD protein might play a dualistic role in mediating not only cell death signaling but other non-apoptotic cellular pathways as well. Here we show that a substantial number of freshly isolated FADD(-/-) peripheral T cells are cycling but are defective in their co-stimulatory response when stimulated. Analysis of several cell cycle proteins shows normal down-regulation of p27 inhibitor but increased levels of p21, decreased levels of cyclin D2, and constitutive activation of several cyclin-dependent kinases in activated T cells. These data suggest that FADD is involved in the regulation of cell cycle machinery in T lymphocytes.

FADD/Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAIL-receptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis ofFADD−/−T cells fromRAG-1−/−reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADD-deficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4−CD8−stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development.

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.

Over-expression of Bcl-2 has been shown to protect thymocytes from apoptosis by many stimuli except strong T cell receptor signals during negative selection. This might be due to Bcl-2 taking on a pro-apoptotic function during negative selection mediated through its BH3 domain, which is exposed upon interaction with the nuclear orphan receptor Nur77. To test this, we generated a T cell-specific transgenic mouse over-expressing Bcl-2 with a mutated BH3 domain. Consistent with our hypothesis, we observed that thymocytes expressing the Bcl-2 BH3 transgene were better protected from apoptosis by TCR stimuli in vitro than those expressing a wild-type transgene. To evaluate TCR-induced apoptosis in vivo, the Bcl-2 BH3 mutant transgenic mice were crossed to F5 and HY TCR transgenic mice. Vibratome cut thymic slices were treated acutely with F5 and HY TCR-specific peptides, circumventing the peripheral cytotoxic effects of in vivo peptide injection. Bcl-2 BH3 mutant transgenic thymocytes were almost completely rescued from peptide-induced apoptosis, but surprisingly, thymocytes expressing the wild-type Bcl-2 transgene were equivalently protected. Thus our results indicate that Bcl-2 has some pro-apoptotic function, but in contrast to previously published data in TCR-transgenic models, we conclude that Bcl-2 over-expression affords substantial protection from apoptosis accompanying negative selection.

SummaryWhile fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes, including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining a BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.