Question: PPAR-RXRs are suppressed by PPAR ligands.

Allosteric Interactions Among Nuclear Hormone Receptors Yield Diverse Response Pathways. Heterodimerization is a prevalent mechanism in eukaryotic transcription factors, exemplified by the 9-cis retinoic acid receptor (RXR) which often partners with various nuclear receptors such as the thyroid hormone receptor (T3R) and retinoic acid receptor (RAR). This raises the question of whether these heterodimeric complexes exhibit responsiveness to dual hormones. To investigate the transcriptional characteristics of each receptor, both individually and within heterodimers, we developed a strategy. Our findings indicate that RXR's inherent binding properties are overshadowed in T3R-RXR and RAR-RXR complexes. Conversely, RXR functions as a non-DNA-binding cofactor when associated with the NGFI-B/Nurr1 orphan receptors. Heterodimerization of RXR with constitutively active NGFI-B/Nurr1 generates a hormone-dependent complex with novel properties. These results suggest that allosteric interactions between heterodimers produce unique complexes, highlighting the importance of allostery in generating diversity within hormone response networks.

Gene regulatory factors encoded by the nuclear genome are essential for mitochondrial biogenesis and function. Some of these factors act exclusively within the mitochondria to regulate the control of mitochondrial transcription, translation, and other functions. Others govern the expression of nuclear genes required for mitochondrial metabolism and organelle biogenesis. The peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family of transcriptional coactivators play a major role in transducing and integrating physiological signals governing metabolism, differentiation, and cell growth to the transcriptional machinery controlling mitochondrial functional capacity. Thus, the PGC-1 coactivators serve as a central component of the transcriptional regulatory circuitry that coordinately controls the energy-generating functions of mitochondria in accordance with the metabolic demands imposed by changing physiological conditions, senescence, and disease.

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. We identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system, and show that these regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. We found that COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Hepatic stellate cells (HSCs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). Vitamin D receptor (VDR) activation in HSCs inhibits liver inflammation and fibrosis. We found that p62/SQSTM1, a protein upregulated in liver parenchymal cells but downregulated in HCC-associated HSCs, negatively controls HSC activation. Total body or HSC-specific p62 ablation potentiates HSCs and enhances inflammation, fibrosis, and HCC progression. p62 directly interacts with VDR and RXR promoting their heterodimerization, which is critical for VDR:RXR target gene recruitment. Loss of p62 in HSCs impairs the repression of fibrosis and inflammation by VDR agonists. This demonstrates that p62 is a negative regulator of liver inflammation and fibrosis through its ability to promote VDR signaling in HSCs, whose activation supports HCC.

Transient activation of estrogen receptors (ER) in the developing brain during a limited perinatal "window of time" is recognized as a key mechanism of defeminization of neural control of reproductive function and sexual behavior. Two major ER isoforms, alpha and beta, are present in neural circuits that govern ovarian cycle and sexual behavior. Using highly selective ER agonists, this study provides the first evidence for distinct contribution of individual ER isoforms to the process of estrogen dependent defeminization. Neonatal activation of the ERalpha in female rats resulted in abrogation of cyclic ovarian activity and female sexual behavior in adulthood. These effects are associated with male-like alterations in the morphology of the anteroventral periventricular (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA), as well as refractoriness to estrogen-mediated induction of sexual receptivity. Exposure to an ERbeta-selective agonist induced persistent estrus and had a strong defeminizing effect on the hypothalamic gonadotropin "surge generator" AVPV. However, neonatal ERbeta activation failed to alter female sexual behavior, responsiveness to estrogens and morphometric features of the behaviorally relevant SDN-POA. Thus, although co-present in several brain regions involved in the control of female reproductive function, ER isoforms convey different, and probably not synergistic, chemical signals in the course of neonatal sex-specific brain organization.

Obesity and type 2 diabetes are associated with increased lipogenesis in the liver. This results in fat accumulation in hepatocytes, a condition known as hepatic steatosis, which is a form of nonalcoholic fatty liver disease (NAFLD), the most common cause of liver dysfunction in the United States. Carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, has emerged as a major player in the development of hepatic steatosis in mice. However, the molecular mechanisms enhancing its transcriptional activity remain largely unknown. In this study, we have identified the histone acetyltransferase (HAT) coactivator p300 and serine/threonine kinase salt-inducible kinase 2 (SIK2) as key upstream regulators of ChREBP activity. In cultured mouse hepatocytes, we showed that glucose-activated p300 acetylated ChREBP on Lys672 and increased its transcriptional activity by enhancing its recruitment to its target gene promoters. SIK2 inhibited p300 HAT activity by direct phosphorylation on Ser89, which in turn decreased ChREBP-mediated lipogenesis in hepatocytes and mice overexpressing SIK2. Moreover, both liver-specific SIK2 knockdown and p300 overexpression resulted in hepatic steatosis, insulin resistance, and inflammation, phenotypes reversed by SIK2/p300 co-overexpression. Finally, in mouse models of type 2 diabetes and obesity, low SIK2 activity was associated with increased p300 HAT activity, ChREBP hyperacetylation, and hepatic steatosis. Our findings suggest that inhibition of hepatic p300 activity may be beneficial for treating hepatic steatosis in obesity and type 2 diabetes and identify SIK2 activators and specific p300 inhibitors as potential targets for pharmaceutical intervention.

The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.

Nonalcoholic fatty liver disease (NAFLD) is a growing worldwide epidemic and an important risk factor for the development of insulin resistance, type 2 diabetes, nonalcoholic steatohepatitis (NASH), and hepatic cellular carcinoma (HCC). Despite the prevalence of NAFLD, lifestyle interventions involving exercise and weight loss are the only accepted treatments for this disease. Over the last decade, numerous experimental compounds have been shown to improve NAFLD in preclinical animal models, and many of these therapeutics have been shown to increase the activity of the cellular energy sensor AMP-activated protein kinase (AMPK). Because AMPK activity is reduced by inflammation, obesity, and diabetes, increasing AMPK activity has been viewed as a viable therapeutic strategy to improve NAFLD. In this review, we propose three primary mechanisms by which AMPK activation may improve NAFLD. In addition, we examine the mechanisms by which AMPK is activated. Finally, we identify 27 studies that have used AMPK activators to reduce NAFLD. Future considerations for studies examining the relationship between AMPK and NAFLD are highlighted.


Question: PPAR-RXRs can be stimulated by PPAR ligands.

Distinct Signaling Pathways Emerge from Allosteric Interactions Between Nuclear Hormone Receptors. The concept of heterodimerization is prevalent among transcription factors in eukaryotes. The retinoid X receptor (RXR) often acts as a heterodimerization partner for various nuclear receptors, such as the thyroid hormone receptor (T3R) and the retinoic acid receptor (RAR). This raises the question of whether these receptor complexes can respond to multiple hormones. To address this, we developed a method to evaluate the transcriptional activity of each receptor, either individually or when forming a heterodimer. Our results indicate that the inherent binding capabilities of RXR are overshadowed when it forms heterodimers with T3R or RAR. Interestingly, RXR demonstrates activity as a non-DNA-binding cofactor in conjunction with the orphan receptors NGFI-B/Nurr1. When RXR heterodimerizes with constitutively active NGFI-B/Nurr1, a new hormone-sensitive complex is formed. These findings suggest that allosteric interactions among heterodimers lead to the creation of complexes with distinct characteristics. We propose that allostery is a key mechanism that contributes to the diversity of hormonal response pathways.

Chronic, nonresolving inflammation is a critical factor in the clinical progression of advanced atherosclerotic lesions. In the normal inflammatory response, resolution is mediated by several agonists, among which is the glucocorticoid-regulated protein called annexin A1. The proresolving actions of annexin A1, which are mediated through its receptor N-formyl peptide receptor 2 (FPR2/ALX), can be mimicked by an amino-terminal peptide encompassing amino acids 2–26 (Ac2-26). Collagen IV (Col IV)–targeted nanoparticles (NPs) containing Ac2-26 were evaluated for their therapeutic effect on chronic, advanced atherosclerosis in fat-fed Ldlr−/− mice. When administered to mice with preexisting lesions, Col IV–Ac2-26 NPs were targeted to lesions and led to a marked improvement in key advanced plaque properties, including an increase in the protective collagen layer overlying lesions (which was associated with a decrease in lesional collagenase activity), suppression of oxidative stress, and a decrease in plaque necrosis. In mice lacking FPR2/ALX in myeloid cells, these improvements were not seen. Thus, administration of a resolution-mediating peptide in a targeted NP activates its receptor on myeloid cells to stabilize advanced atherosclerotic lesions. These findings support the concept that defective inflammation resolution plays a role in advanced atherosclerosis, and suggest a new form of therapy.

1. The liver is the target organ for the lipid-regulating effect of rosuvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and liver-selective uptake of this drug is therefore a desirable property. The uptake kinetics of rosuvastatin were investigated and compared with those of pravastatin using isolated rat hepatocytes. 2. Uptake for both drugs involved both active transport and passive diffusion processes. The Michaelis constant (K(m)) of uptake rate for rosuvastatin (9.17 micro M) was approximately half that for pravastatin (16.5 micro M). However, the maximum uptake rate (V(max)) and carrier-mediated uptake clearance (V(max)/K(m)) of rosuvastatin were significantly (p < 0.01) greater than those of pravastatin, and a larger contribution of carrier-mediated uptake clearance to total uptake clearance was shown for rosuvastatin (contribution ratio 0.903 versus pravastatin 0.654). 3. Sodium and chloride ions did not play a significant role in the uptake of rosuvastatin and pravastatin, but the uptake of both drugs was inhibited both by depletion of cellular ATP and by organic anions such as bromosulfophthalein. 4. Rosuvastatin competitively inhibited the uptake of pravastatin, with an inhibition constant (K(i)) (2.75 micro M) relatively similar to its K(m). 5. The results suggest that an organic anion transport protein is the main mediator of the hepatic uptake of rosuvastatin and pravastatin, which occurs in an ATP-dependent manner. Our results indicated that rosuvastatin was taken up by the hepatocytes via the same transport systems as pravastatin, but with a greater affinity and efficiency than pravastatin.

We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)

Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor against oxidative stress and neurodegenerative disorders. Phenylethanoid glycosides (PhGs; salidroside, acteoside, isoacteoside, and echinacoside) exhibit antioxidant and neuroprotective bioactivities. This study was performed to investigate the neuroprotective effect and molecular mechanism of PhGs. PhGs pretreatment significantly suppressed H₂O₂-induced cytotoxicity in PC12 cells by triggering the nuclear translocation of Nrf2 and reversing the downregulated protein expression of heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate cysteine ligase-catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM). Nrf2 siRNA or HO-1 inhibitor zinc protoporphyrin (ZnPP) reduced the neuroprotective effect. PhGs showed potential interaction with the Nrf2 binding site in Kelch-like ECH-association protein 1 (Keap1). This result may support the hypothesis that PhGs are activators of Nrf2. We demonstrated the potential binding between PhGs and the Keap1-activated Nrf2/ARE pathway, and that PhGs with more glycosides had enhanced effects.

Bile acids are recognized as metabolic modulators. The present study was aimed at evaluating the effects of a potent Asbt inhibitor (264W94), which blocks intestinal absorption of bile acids, on glucose homeostasis in Zucker Diabetic Fatty (ZDF) rats. Oral administration of 264W94 for two wk increased fecal bile acid concentrations and elevated non-fasting plasma total Glp-1. Treatment of 264W94 significantly decreased HbA1c and glucose, and prevented the drop of insulin levels typical of ZDF rats in a dose-dependent manner. An oral glucose tolerance test revealed up to two-fold increase in plasma total Glp-1 and three-fold increase in insulin in 264W94 treated ZDF rats at doses sufficient to achieve glycemic control. Tissue mRNA analysis indicated a decrease in farnesoid X receptor (Fxr) activation in small intestines and the liver but co-administration of a Fxr agonist (GW4064) did not attenuate 264W94 induced glucose lowering effects. In summary, our results demonstrate that inhibition of Asbt increases bile acids in the distal intestine, promotes Glp-1 release and may offer a new therapeutic strategy for type 2 diabetes mellitus.

Gene regulatory factors encoded by the nuclear genome are essential for mitochondrial biogenesis and function. Some of these factors act exclusively within the mitochondria to regulate the control of mitochondrial transcription, translation, and other functions. Others govern the expression of nuclear genes required for mitochondrial metabolism and organelle biogenesis. The peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family of transcriptional coactivators play a major role in transducing and integrating physiological signals governing metabolism, differentiation, and cell growth to the transcriptional machinery controlling mitochondrial functional capacity. Thus, the PGC-1 coactivators serve as a central component of the transcriptional regulatory circuitry that coordinately controls the energy-generating functions of mitochondria in accordance with the metabolic demands imposed by changing physiological conditions, senescence, and disease.

Anabolic steroids have been widely used in the beef cattle industry for over 50 years as safe and effective growth-promoting agents, and today, more than 90% of all feedlot cattle in the US receive some type of steroidal implant during their lifetime (NAHMS, USDA 2000). A list of approval of growth promotants by the FDA is shown in Table 1. Generally, implants have been shown to increase growth rate 8 to 28%, improve feed efficiency 5 to 20%, and enhance lean tissue mass of the carcass 3 to 10% (Duckett and Owens, 1997). This improvement in production efficiency has multiple benefits: 1) it clearly and dramatically reduces production costs by reducing the amount of feed required per unit of gain (Avery and Avery, 2007); 2) it reduces the amount of land necessary to produce equivalent amounts of food for consumers; 3) it limits the production of greenhouse gases by reducing the number of animals required to produce equivalent amounts of beef (Avery and Avery, 2007); and 4) it extends cost savings to consumers by providing a year-round, affordable supply of beef at reduced prices (Lawrence and Ibarburu, 2006). Since animal products contribute significantly to the total caloric and nutrient intake in the human population, altering the composition of growth toward more lean tissue and less adipose tissue results in a healthier product with fewer calories that still is rich in beneficial nutrients. In addition to steroidal implants, a newer class of orally active growth promotants, known as b-adrenergic agonists (BAA; Ractopamine-HCl; 2003 and Zilpaterol-HCl; 2006), have been approved for use in finishing beef cattle in the last decade. These products provide similar production benefits as steroidal implants, but differ in application and mode of action. Beta-adrenergic agonists are fed during the last 20 to 42 days of the cattle finishing period, depending on the specific product. The purpose of this paper is to explain how anabolic implants and BAA work and their benefits to beef cattle production.

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.

Objective: To investigate the regulation of CD4+CD25+ Regulatory T cells (Tregs) on pro-inflammatory adhesion molecules, Krüppel-Like Factor-2 (KLF-2) and its downstream transcriptional targets in human umbilical vein endothelial cells (HUVECs) impaired by ox-LDL and the mechanisms of it. Methods and results: HUVECs were cultured in the continuous presence of ox-LDL(0 mg/L,25 mg/L,50 mg/L,100 mg/L) for 4, 6, 12 and 24 hours to allow identification of early-and late-induced genes, respectively, whereas non-stimulated controls were taken at 0 hours. The expression of pro-inflammatory adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, KLF-2 and its target genes eNOS, PAI-1 were determined by real time RT-PCR and/or western-blot analysis. Expression of pro-inflammatory adhesion molecules, KLF-2, eNOS and PAI-1 in HUVEC cultured alone or with anti-CD3 mAbs activated Tregs, followed by addition of ox-LDL (50 mg/L) for 6 hours, are compared to expression levels in control cultures. Ox-LDL treated HUVECs increased pro-inflammatory adhesion molecules expression, as well as increased PAI-1 but decreased eNOS expression accompanied with significant downregulating of KLF-2 at a dose and time dependent manner. Furthermore, ox-LDL increased pro-inflammatory adhesion molecules but inhibited KLF2 expression was reversed by addition of Tregs. Small interfering RNA reduced endogenous KLF-2 expression and partly reversed the suppressive effect of Tregs on HUVECs activation, which strongly implicate KLF-2 as a transcriptional regulator of the Tregs-mediated effects in endothelial cells. Mechanism studies reveal that Treg-mediated KLF2 expression in HUVECs impaired by ox-LDL requires cell contact as well as soluble factors. Conclusions: Tregs could protect endothelial function that is largely dependent on KLF2 and its downstream transcriptional targets regulation involving cell-to-cell contact and soluble factors.


Question: Engaging in six months of physical exercise enhances cognitive performance.

Impact of Physical Exercise on Cognitive Performance in Older Adults with Memory Concerns: A Randomized Controlled Study. BACKGROUND Numerous observational studies have indicated that regular physical activity may lower the risk of cognitive decline. However, there is a scarcity of evidence from randomized controlled trials. OBJECTIVE This study aimed to assess whether physical exercise can mitigate cognitive decline in older adults with memory impairment but not dementia. DESIGN AND LOCATION A randomized controlled trial was conducted in Perth, Western Australia, between 2004 and 2007, involving a 24-week physical activity program. Assessors of cognitive function were blinded to participant group allocation. PARTICIPANTS We enrolled volunteers who reported memory issues but did not meet the criteria for dementia. A total of 311 individuals aged 50 and above were screened for eligibility; 89 were ineligible, and 52 declined participation. Out of 170 randomized participants, 138 completed the 18-month assessment. INTERVENTION Participants were randomly assigned to either an education and usual care group or a 24-week home-based physical activity program. MAIN OUTCOME MEASURE The change in Alzheimer Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) scores (range: 0-70) over an 18-month period. RESULTS In an intent-to-treat analysis, participants in the intervention group experienced a 0.26 point improvement (95% confidence interval: -0.89 to 0.54), while those in the usual care group had a 1.04 point decline (95% confidence interval: 0.32 to 1.82) in ADAS-Cog scores at the end of the intervention. The absolute difference between the intervention and control groups was -1.3 points (95% confidence interval: -2.38 to -0.22) at the end of the intervention. At the 18-month follow-up, participants in the intervention group showed a 0.73 point improvement (95% confidence interval: -1.27 to 0.03), whereas those in the usual care group had a 0.04 point improvement (95% confidence interval: -0.46 to 0.88) in ADAS-Cog scores. There were also slight improvements in word list delayed recall and Clinical Dementia Rating sum of boxes, whereas word list total immediate recall, digit symbol coding, verbal flu

CONTEXT Although acute hypoglycemia may be associated with cognitive impairment in children with type 1 diabetes, no studies to date have evaluated whether hypoglycemia is a risk factor for dementia in older patients with type 2 diabetes. OBJECTIVE To determine if hypoglycemic episodes severe enough to require hospitalization are associated with an increased risk of dementia in a population of older patients with type 2 diabetes followed up for 27 years. DESIGN, SETTING, AND PATIENTS A longitudinal cohort study from 1980-2007 of 16,667 patients with a mean age of 65 years and type 2 diabetes who are members of an integrated health care delivery system in northern California. MAIN OUTCOME MEASURE Hypoglycemic events from 1980-2002 were collected and reviewed using hospital discharge and emergency department diagnoses. Cohort members with no prior diagnoses of dementia, mild cognitive impairment, or general memory complaints as of January 1, 2003, were followed up for a dementia diagnosis through January 15, 2007. Dementia risk was examined using Cox proportional hazard regression models, adjusted for age, sex, race/ethnicity, education, body mass index, duration of diabetes, 7-year mean glycated hemoglobin, diabetes treatment, duration of insulin use, hyperlipidemia, hypertension, cardiovascular disease, stroke, transient cerebral ischemia, and end-stage renal disease. RESULTS At least 1 episode of hypoglycemia was diagnosed in 1465 patients (8.8%) and dementia was diagnosed in 1822 patients (11%) during follow-up; 250 patients had both dementia and at least 1 episode of hypoglycemia (16.95%). Compared with patients with no hypoglycemia, patients with single or multiple episodes had a graded increase in risk with fully adjusted hazard ratios (HRs): for 1 episode (HR, 1.26; 95% confidence interval [CI], 1.10-1.49); 2 episodes (HR, 1.80; 95% CI, 1.37-2.36); and 3 or more episodes (HR, 1.94; 95% CI, 1.42-2.64). The attributable risk of dementia between individuals with and without a history of hypoglycemia was 2.39% per year (95% CI, 1.72%-3.01%). Results were not attenuated when medical utilization rates, length of health plan membership, or time since initial diabetes diagnosis were added to the model. When examining emergency department admissions for hypoglycemia for association with risk of dementia (535 episodes), results were similar (compared with patients with 0 episodes) with fully adjusted HRs: for 1 episode (HR, 1.42; 95% CI, 1.12-1.78) and for 2 or more episodes (HR, 2.36; 95% CI, 1.57-3.55). CONCLUSIONS Among older patients with type 2 diabetes, a history of severe hypoglycemic episodes was associated with a greater risk of dementia. Whether minor hypoglycemic episodes increase risk of dementia is unknown.

Objective: To examine different health outcomes that are associated with specific lifestyle and genetic factors. Materials and methods: From March 2004 to April 2006, a sample of employees from three different health and academic institutions, as well as their family members, were enrolled in the study after providing informed consent. At baseline and follow-up (2010-2013), participants completed a self-administered questionnaire, a physical examination, and provided blood samples. Results: A total of 10 729 participants aged 6 to 94 years were recruited at baseline. Of these, 70% were females, and 50% were from the Mexican Social Security Institute. Nearly 42% of the adults in the sample were overweight, while 20% were obese. Conclusion: Our study can offer new insights into disease mechanisms and prevention through the analysis of risk factor information in a large sample of Mexicans.

OBJECTIVE To determine whether exposure to low doses of ionising radiation in infancy affects cognitive function in adulthood. DESIGN Population based cohort study. SETTING Sweden. PARTICIPANTS 3094 men who had received radiation for cutaneous haemangioma before age 18 months during 1930-59. MAIN OUTCOME MEASURES Radiation dose to frontal and posterior parts of the brain, and association between dose and intellectual capacity at age 18 or 19 years based on cognitive tests (learning ability, logical reasoning, spatial recognition) and high school attendance. RESULTS The proportion of boys who attended high school decreased with increasing doses of radiation to both the frontal and the posterior parts of the brain from about 32% among those not exposed to around 17% in those who received > 250 mGy. For the frontal dose, the multivariate odds ratio was 0.47 (95% confidence interval 0.26 to 0.85, P for trend 0.0003) and for the posterior dose it was 0.59 (0.23 to 1.47, 0.0005). A negative dose-response relation was also evident for the three cognitive tests for learning ability and logical reasoning but not for the test of spatial recognition. CONCLUSIONS Low doses of ionising radiation to the brain in infancy influence cognitive abilities in adulthood.

The subjective and behavioural responses of 20 healthy volunteers taking droperidol 5 mg as part of a cognitive challenge programme were catalogued. Some form of akathisia was universally experienced. Half of the subjects were dysphoric, but there appeared to be a number of different inputs to their dysphoria and a range of other effects were noted, including sedation, dissociative experiences, alterations in sensation and subtle changes in physiognomy. The duration of these effects varied from a few hours to over a week. In the acute phase, insight as to the origin of what was happening was mixed. The results have implications for the interpretation of cognitive challenge tests, the nature of akathisia, clinical therapeutics and drug development. © 1998 John Wiley & Sons, Ltd.

The concept of frailty as a medically distinct syndrome has evolved based on the clinical experience of geriatricians and is clinically well recognizable. Frailty is a nonspecific state of vulnerability, which reflects multisystem physiological change. These changes underlying frailty do not always achieve disease status, so some people, usually very elderly, are frail without a specific life threatening illness. Current thinking is that not only physical but also psychological, cognitive and social factors contribute to this syndrome and need to be taken into account in its definition and treatment. Together, these signs and symptoms seem to reflect a reduced functional reserve and consequent decrease in adaptation (resilience) to any sort of stressor and perhaps even in the absence of extrinsic stressors. The overall consequence is that frail elderly are at higher risk for accelerated physical and cognitive decline, disability and death. All these characteristics associated with frailty can easily be applied to the definition and characterization of the aging process per se and there is little consensus in the literature concerning the physiological/biological pathways associated with or determining frailty. It is probably true to say that a consensus view would implicate heightened chronic systemic inflammation as a major contributor to frailty. This review will focus on the relationship between aging, frailty and age-related diseases, and will highlight possible interventions to reduce the occurrence and effects of frailty in elderly people.

BACKGROUND The main associations of body-mass index (BMI) with overall and cause-specific mortality can best be assessed by long-term prospective follow-up of large numbers of people. The Prospective Studies Collaboration aimed to investigate these associations by sharing data from many studies. METHODS Collaborative analyses were undertaken of baseline BMI versus mortality in 57 prospective studies with 894 576 participants, mostly in western Europe and North America (61% [n=541 452] male, mean recruitment age 46 [SD 11] years, median recruitment year 1979 [IQR 1975-85], mean BMI 25 [SD 4] kg/m(2)). The analyses were adjusted for age, sex, smoking status, and study. To limit reverse causality, the first 5 years of follow-up were excluded, leaving 66 552 deaths of known cause during a mean of 8 (SD 6) further years of follow-up (mean age at death 67 [SD 10] years): 30 416 vascular; 2070 diabetic, renal or hepatic; 22 592 neoplastic; 3770 respiratory; 7704 other. FINDINGS In both sexes, mortality was lowest at about 22.5-25 kg/m(2). Above this range, positive associations were recorded for several specific causes and inverse associations for none, the absolute excess risks for higher BMI and smoking were roughly additive, and each 5 kg/m(2) higher BMI was on average associated with about 30% higher overall mortality (hazard ratio per 5 kg/m(2) [HR] 1.29 [95% CI 1.27-1.32]): 40% for vascular mortality (HR 1.41 [1.37-1.45]); 60-120% for diabetic, renal, and hepatic mortality (HRs 2.16 [1.89-2.46], 1.59 [1.27-1.99], and 1.82 [1.59-2.09], respectively); 10% for neoplastic mortality (HR 1.10 [1.06-1.15]); and 20% for respiratory and for all other mortality (HRs 1.20 [1.07-1.34] and 1.20 [1.16-1.25], respectively). Below the range 22.5-25 kg/m(2), BMI was associated inversely with overall mortality, mainly because of strong inverse associations with respiratory disease and lung cancer. These inverse associations were much stronger for smokers than for non-smokers, despite cigarette consumption per smoker varying little with BMI. INTERPRETATION Although other anthropometric measures (eg, waist circumference, waist-to-hip ratio) could well add extra information to BMI, and BMI to them, BMI is in itself a strong predictor of overall mortality both above and below the apparent optimum of about 22.5-25 kg/m(2). The progressive excess mortality above this range is due mainly to vascular disease and is probably largely causal. At 30-35 kg/m(2), median survival is reduced by 2-4 years; at 40-45 kg/m(2), it is reduced by 8-10 years (which is comparable with the effects of smoking). The definite excess mortality below 22.5 kg/m(2) is due mainly to smoking-related diseases, and is not fully explained.

BACKGROUND The objective of this study was to evaluate the effectiveness of a brief, 4-session cognitive behavioral, group psychotherapy for binge eating among bariatric surgery candidates at an academic medical center. Binge eating behaviors have been linked to poorer outcomes among bariatric surgery patients, and binge eating disorder have be considered a contraindication in surgery programs, some of which have mandated preoperative binge eating treatment. However, no previous studies have examined whether a preoperative binge eating intervention could successfully reduce binge eating behaviors among severely obese bariatric surgery candidates. METHODS A total of 243 bariatric surgery candidates completed a brief cognitive behavioral group treatment for binge eating behaviors and were administered the Binge Eating Scale and reported the number of weekly binge eating episodes at the initial psychological evaluation and again after the group sessions. The study used a pre-post intervention design. RESULTS The results suggested significant reductions in both binge eating behaviors and cognitions and binge eating episodes after the group intervention. The intervention's effectiveness did not differ according to gender or ethnicity (black versus white). CONCLUSION A brief cognitive behavioral intervention can reduce binge eating behaviors among bariatric surgery candidates. Given the potential influence of binge eating on outcomes, bariatric surgery programs could benefit by treating binge eating before surgery.

CONTEXT Limited information exists regarding the role of left ventricular function in predicting exercise capacity and impact on age- and sex-related differences. OBJECTIVES To determine the impact of measures of cardiac function assessed by echocardiography on exercise capacity and to determine if these associations are modified by sex or advancing age. DESIGN Cross-sectional study of patients undergoing exercise echocardiography with routine measurements of left ventricular systolic and diastolic function by 2-dimensional and Doppler techniques. Analyses were conducted to determine the strongest correlates of exercise capacity and the age and sex interactions of these variables with exercise capacity. SETTING Large tertiary referral center in Rochester, Minnesota, in 2006. PARTICIPANTS Patients undergoing exercise echocardiography using the Bruce protocol (N = 2867). Patients with echocardiographic evidence of exercise-induced ischemia, ejection fractions lower than 50%, or significant valvular heart disease were excluded. MAIN OUTCOME MEASURE Exercise capacity in metabolic equivalents (METs). RESULTS Diastolic dysfunction was strongly and inversely associated with exercise capacity. Compared with normal function, after multivariate adjustment, those with moderate/severe resting diastolic dysfunction (-1.30 METs; 95% confidence interval [CI], -1.52 to -0.99; P < .001) and mild resting diastolic dysfunction (-0.70 METs; 95% CI, -0.88 to -0.46; P < .001) had substantially lower exercise capacity. Variation of left ventricular systolic function within the normal range was not associated with exercise capacity. Left ventricular filling pressures measured by resting E/e' of 15 or greater (-0.41 METs; 95% CI, -0.70 to -0.11; P = .007) or postexercise E/e' of 15 or greater (-0.41 METs; 95% CI, -0.71 to -0.11; P = .007) were similarly associated with a reduction in exercise capacity, each in separate multivariate analyses. Individuals with impaired relaxation (mild dysfunction) or resting E/e' of 15 or greater had a progressive increase in the magnitude of reduction in exercise capacity with advancing age (P < .001 and P = .02, respectively). Other independent correlates of exercise capacity were age (unstandardized beta coefficient, -0.85 METs; 95% CI, -0.92 to -0.77, per 10-year increment; P < .001), female sex (-1.98 METs; 95% CI, -2.15 to -1.84; P < .001), and body mass index greater than 30 (-1.24 METs; 95% CI, -1.41 to -1.10; P < .001). CONCLUSION In this large cross-sectional study of those referred for exercise echocardiography and not limited by ischemia, abnormalities of left ventricular diastolic function were independently associated with exercise capacity.

OBJECTIVE To demonstrate the relation of exercise capacity and BMI to mortality in a population of male veterans with type 2 diabetes. RESEARCH DESIGN AND METHODS After excluding two underweight patients (BMI <18.5 kg/m2), the study population comprised 831 consecutive patients with type 2 diabetes (mean age 61 +/- 9 years) referred for exercise testing for clinical reasons between 1995 and 2006. Exercise capacity was determined from a maximal exercise test and measured in metabolic equivalents (METs). Patients were classified both according to BMI category (18.5-24.9, 25.0-29.9, and > or =30 kg/m2) and by exercise capacity (<5.0 or > or =5.0 maximal METs). The association among exercise capacity, BMI, other clinical variables, and all-cause mortality was assessed by Cox proportional hazards. Study participants were followed for mortality up to 30 June 2006. RESULTS During a mean follow-up of 4.8 +/- 3.0 years, 112 patients died, for an average annual mortality rate of 2.2%. Each 1-MET increase in exercise capacity conferred a 10% survival benefit (hazard ratio 0.90 [95% CI 0.82-0.98]; P = 0.01), but BMI was not significantly associated with mortality. After adjustment for age, ethnicity, examination year, BMI, presence of cardiovascular disease (CVD), and CVD risk factors, diabetic patients achieving <5 maximal METs were 70% more likely to die (1.70 [1.13-2.54]) than those achieving > or =5 maximal METs. CONCLUSIONS There was a strong inverse association between exercise capacity and mortality in this cohort of men with documented diabetes, and this relationship was independent of BMI.

BACKGROUND The increasing prevalence of overweight and obesity underscores the need for evidence-based, easily disseminable interventions for weight management that can be delivered on a population basis. The Transtheoretical Model (TTM) offers a promising theoretical framework for multiple behavior weight management interventions. METHODS Overweight or obese adults (BMI 25-39.9; n=1277) were randomized to no-treatment control or home-based, stage-matched multiple behavior interventions for up to three behaviors related to weight management at 0, 3, 6, and 9 months. All participants were re-assessed at 6, 12, and 24 months. RESULTS Significant treatment effects were found for healthy eating (47.5% versus 34.3%), exercise (44.90% versus 38.10%), managing emotional distress (49.7% versus 30.30%), and untreated fruit and vegetable intake (48.5% versus 39.0%) progressing to Action/Maintenance at 24 months. The groups differed on weight lost at 24 months. Co-variation of behavior change occurred and was much more pronounced in the treatment group, where individuals progressing to Action/Maintenance for a single behavior were 2.5-5 times more likely to make progress on another behavior. The impact of the multiple behavior intervention was more than three times that of single behavior interventions. CONCLUSIONS This study demonstrates the ability of TTM-based tailored feedback to improve healthy eating, exercise, managing emotional distress, and weight on a population basis. The treatment produced a high level of population impact that future multiple behavior interventions can seek to surpass.


Question: Individuals in steady relationships experience a quicker development from HIV to AIDS.

Stable Relationships and Improved Clinical Outcomes in HIV-infected Individuals on Antiretroviral Therapy: Evidence from the Swiss HIV Cohort Study. AIMS This study aimed to investigate the correlation between stable relationships and clinical outcomes in HIV-positive patients undergoing highly active antiretroviral therapy (HAART). METHODOLOGY A prospective cohort study was conducted involving adult HIV patients (Swiss HIV cohort study) across seven outpatient clinics in Switzerland. The analysis included 3736 patients who initiated HAART before 2002 (median age 36 years, 29% female, median follow-up 3.6 years). KEY OUTCOME METRICS Time to development of AIDS or death (primary endpoint), death alone, CD4 cell count increase of 50 or 100 cells above baseline, optimal viral suppression (viral load below 400 copies/ml), and viral rebound. RESULTS Over the follow-up period, 2985 (80%) participants reported having a stable relationship at least once. At the start of HAART, 52% (545/1042) of participants were in a stable relationship; this decreased to 46% (190/412) after five years. Stratified analysis by prior antiretroviral therapy and clinical stage when initiating HAART (US CDC groups A, B, or C) showed that individuals with a stable relationship had an adjusted hazard ratio of 0.79 (95% CI 0.63 to 0.98) for progressing to AIDS or death compared to those without. Additional adjusted hazard ratios were 0.59 (0.44 to 0.79) for progressing to death, 1.15 (1.06 to 1.24) for a CD4 cell count increase of 100 cells/microl or more, and 1.06 (0.98 to 1.14) for optimal viral suppression. CONCLUSION A stable relationship is linked to a reduced pace of progression to AIDS or death in HIV-infected individuals on HAART.

OBJECTIVE This study compared, in treatment and control groups, the phenomena of coaction, which is the probability that taking effective action on one behavior is related to taking effective action on a second behavior. METHODS Pooled data from three randomized trials of Transtheoretical Model (TTM) tailored interventions (n=9461), completed in the U.S. in 1999, were analyzed to assess coaction in three behavior pairs (diet and sun protection, diet and smoking, and sun protection and smoking). Odds ratios (ORs) compared the likelihood of taking action on a second behavior compared to taking action on only one behavior. RESULTS Across behavior pairs, at 12 and 24 months, the ORs for the treatment group were greater on an absolute basis than for the control group, with two being significant. The combined ORs at 12 and 24 months, respectively, were 1.63 and 1.85 for treatment and 1.20 and 1.10 for control. CONCLUSIONS The results of this study with addictive, energy balance and appearance-related behaviors were consistent with results found in three studies applying TTM tailoring to energy balance behaviors. Across studies, there was more coaction within the treatment group. Future research should identify predictors of coaction in more multiple behavior change interventions.

Injection drug users (IDUs) are at increased risk for HIV, viral hepatitis, and tuberculosis, and making up more than a quarter of the incarcerated population in the United States. Methadone maintenance treatment of opiate addiction is highly effective at reducing drug use, drug-related criminal activity, and risk of HIV transmission. Recently released inmates are at particularly high risk for overdose and disease transmission. Linkage to methadone treatment immediately upon release from incarceration is a promising opportunity to combat disease transmission, facilitate reentry into the community, and reduce recidivism.

CONTEXT More than 1.5 million US adults use stimulants and other medications labeled for treatment of attention-deficit/hyperactivity disorder (ADHD). These agents can increase heart rate and blood pressure, raising concerns about their cardiovascular safety. OBJECTIVE To examine whether current use of medications prescribed primarily to treat ADHD is associated with increased risk of serious cardiovascular events in young and middle-aged adults. DESIGN, SETTING, AND PARTICIPANTS Retrospective, population-based cohort study using electronic health care records from 4 study sites (OptumInsight Epidemiology, Tennessee Medicaid, Kaiser Permanente California, and the HMO Research Network), starting in 1986 at 1 site and ending in 2005 at all sites, with additional covariate assessment using 2007 survey data. Participants were adults aged 25 through 64 years with dispensed prescriptions for methylphenidate, amphetamine, or atomoxetine at baseline. Each medication user (n = 150,359) was matched to 2 nonusers on study site, birth year, sex, and calendar year (443,198 total users and nonusers). MAIN OUTCOME MEASURES Serious cardiovascular events, including myocardial infarction (MI), sudden cardiac death (SCD), or stroke, with comparison between current or new users and remote users to account for potential healthy-user bias. RESULTS During 806,182 person-years of follow-up (median, 1.3 years per person), 1357 cases of MI, 296 cases of SCD, and 575 cases of stroke occurred. There were 107,322 person-years of current use (median, 0.33 years), with a crude incidence per 1000 person-years of 1.34 (95% CI, 1.14-1.57) for MI, 0.30 (95% CI, 0.20-0.42) for SCD, and 0.56 (95% CI, 0.43-0.72) for stroke. The multivariable-adjusted rate ratio (RR) of serious cardiovascular events for current use vs nonuse of ADHD medications was 0.83 (95% CI, 0.72-0.96). Among new users of ADHD medications, the adjusted RR was 0.77 (95% CI, 0.63-0.94). The adjusted RR for current use vs remote use was 1.03 (95% CI, 0.86-1.24); for new use vs remote use, the adjusted RR was 1.02 (95% CI, 0.82-1.28); the upper limit of 1.28 corresponds to an additional 0.19 events per 1000 person-years at ages 25-44 years and 0.77 events per 1000 person-years at ages 45-64 years. CONCLUSIONS Among young and middle-aged adults, current or new use of ADHD medications, compared with nonuse or remote use, was not associated with an increased risk of serious cardiovascular events. Apparent protective associations likely represent healthy-user bias.

CONTEXT The epidemic of heart failure has yet to be fully investigated, and data on incidence, survival, and sex-specific temporal trends in community-based populations are limited. OBJECTIVE To test the hypothesis that the incidence of heart failure has declined and survival after heart failure diagnosis has improved over time but that secular trends have diverged by sex. DESIGN, SETTING, AND PARTICIPANTS Population-based cohort study using the resources of the Rochester Epidemiology Project conducted in Olmsted County, Minnesota. Patients were 4537 Olmsted County residents (57% women; mean [SD] age, 74 [14] years) with a diagnosis of heart failure between 1979 and 2000. Framingham criteria and clinical criteria were used to validate the diagnosis   MAIN OUTCOME MEASURES Incidence of heart failure and survival after heart failure diagnosis. RESULTS The incidence of heart failure was higher among men (378/100 000 persons; 95% confidence interval [CI], 361-395 for men; 289/100 000 persons; 95% CI, 277-300 for women) and did not change over time among men or women. After a mean follow-up of 4.2 years (range, 0-23.8 years), 3347 deaths occurred, including 1930 among women and 1417 among men. Survival after heart failure diagnosis was worse among men than women (relative risk, 1.33; 95% CI, 1.24-1.43) but overall improved over time (5-year age-adjusted survival, 43% in 1979-1984 vs 52% in 1996-2000, P<.001). However, men and younger persons experienced larger survival gains, contrasting with less or no improvement for women and elderly persons. CONCLUSION In this community-based cohort, the incidence of heart failure has not declined during 2 decades, but survival after onset of heart failure has increased overall, with less improvement among women and elderly persons.

BACKGROUND AND AIMS We adopted the twin method to disentangle the genetic and environmental components of susceptibility to coeliac disease (CD). We estimated disease concordance rate by zygosity and HLA genotypes, discordance times, progression rates to disease, and heritability. METHODS We crosslinked the Italian Twin Registry with the membership lists of the Italian Coeliac Disease Association and recruited 23 monozygotic (MZ) and 50 dizygotic (DZ) twin pairs with at least one affected member. Zygosity was assigned by DNA fingerprinting, and HLA-DQ and DR alleles were genotyped. Disease status was ascertained by antiendomysial, anti-human tissue transglutaminase antibodies, and bowel biopsy. RESULTS Concordance was significantly higher in MZ (83.3% probandwise, 71.4% pairwise) than in DZ (16.7% probandwise, 9.1% pairwise) pairs. Concordance was not affected by sex or HLA genotype of the co-twin and being MZ was significantly associated with the occurrence of CD (Cox adjusted hazard ratio 14.3 (95% confidence interval 4.0-50.3)). In 90% of concordant pairs the discordance time was <or=2 years. MZ and DZ co-twins had 70% and 9% cumulative probability of having symptomatic or silent forms of CD, respectively, within five years. Under ACE (additive genetic, common, and unshared environmental factors) models, with CD population prevalences of 1/91 and 1/1000, heritability estimates were 87% and 57%, respectively. CONCLUSION MZ pairs have a high probability of being concordant, regardless of sex or HLA genotype. Most of the affected co-twins receive a diagnosis within two years. A remarkable proportion of phenotypic variance is due to genetic factors.

As the global incidence of HIV exceeds 2 million new infections annually, effective interventions to decrease HIV transmission are needed. Randomized, placebo-controlled studies have demonstrated that daily oral antiretroviral pre-exposure prophylaxis (PrEP) with a fixed-dose combination tablet containing tenofovir disoproxil fumarate and emtricitabine can significantly reduce HIV incidence among diverse at-risk populations. In these studies, the efficacy of PrEP was correlated with levels of adherence. Official guidelines recommend provision of PrEP to people at greatest risk of HIV acquisition, and demonstration projects suggest that high levels of uptake and adherence are possible outside of controlled studies. However, several potential barriers to implementing PrEP remain. These challenges include low awareness and utilization of PrEP by at-risk individuals, uncertainty about adherence in ‘real-world’ settings, the majority of healthcare providers being untrained in PrEP provision, limited data about potential adverse effects from long-term use of tenofovir–emtricitabine, high costs of PrEP medications, and stigma associated with PrEP use and the behaviors that would warrant PrEP. Innovative pharmacologic chemoprophylactic approaches could provide solutions to some of these challenges. Less-than-daily oral dosing regimens and long-acting injectable medications could reduce pill burdens and facilitate adherence, and local delivery of PrEP medications to genital compartments via gels, rings and films may limit systemic drug exposure and potential toxicities. As the portfolio of chemoprophylactic agents and delivery systems expands to meet the diverse sexual health needs and product preferences of individuals who may benefit from PrEP, it is hoped that antiretroviral chemoprophylaxis could become an acceptable, feasible, and highly effective addition to existing HIV prevention strategies.

In humans, apolipoprotein E (apoE) is a polymorphic multifunctional protein.1 It is coded by three alleles (e2, e3, e4) of a modulator gene (level, variability, and susceptibility gene) at the apoE locus on chromosome 19, determining six apoE genotypes and plasma phenotypes. Its pleiotropic effects are exerted on plasma lipoprotein metabolism, coagulation, oxidative processes, macrophage, glial cell and neuronal cell homeostasis, adrenal function, central nervous system physiology, inflammation, and cell proliferation.2,3 ApoE polymorphism modulates susceptibility to many diseases. It is, however, particularly notorious for its role in neurodegenerative disorders4 and atherosclerotic arterial disease.5,6 The e4 allele (phenotypes E4/4 and E4/3) that is associated with higher low density lipoprotein cholesterol (LDL-C) is considered proatherogenic, whereas the presence of the e2 allele (E3/2, E2/2), being associated with lower LDL-C levels, is deemed to have the opposite effect (although it may be associated with increased plasma triglycerides and lipoprotein remnants). This simple equation, however, is an oversimplification because these properties are subject to many environmental and genetic influences. ApoE has allele- and gender-dependent effects on reverse cholesterol transport, platelet aggregation, and oxidative processes that are likely to affect the overall atherogenic potential ascribed to modulation of lipoprotein metabolism.2,3,6 Notwithstanding the context dependency, a recent meta-analysis fully supports the presence of the e4 allele as a significant risk factor for coronary artery disease.7 Several mechanisms have been evoked to link apoE with atherosclerosis, but the relationship is not fully unraveled in humans. Nevertheless, some apoE mimetic peptides that promote LDL clearance are currently tested in animals for potential clinical applications.8,9  See page 436   The situation is relatively simpler in animals. The mouse model has been prominently useful to test mechanisms …

Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis. Although incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known cross-talk between the brain and immune system includes the hypothalamic-pituitary-adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic-adrenal-medullary axis, which controls stress-induced catecholamine release in support of the fight-or-flight reflex. It remains unknown, however, whether chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive hematopoietic progenitors, giving rise to higher levels of disease-promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the β3-adrenergic receptor. Consequently, hematopoietic stem cell proliferation was elevated, leading to an increased output of neutrophils and inflammatory monocytes. When atherosclerosis-prone Apoe(-/-) mice were subjected to chronic stress, accelerated hematopoiesis promoted plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

Multiple sclerosis (MS), a chronic inflammatory demyelina-ting and degenerative disease of the central nervous system, is a frequent cause of neurological disability in young adults. Female predominance has increased over the last decades. Although female gender carries a higher risk of developing relapsing remitting MS, being female and at child-bearing age also appears to provide some protection against cognitive decline and against progressive onset MS, an adverse predictive factor when considering long-term disability in MS. The risk of MS in women has been associated with an earlier age at menarche. In most studies, parity did not impact MS risk. However, the recently published association of higher parity and offspring number with a reduced risk of a first demyelinating event suggests a potential suppressive effect. Pregnancy in MS patients has been associated with a reduced relapse rate and a reduction of neurological symptoms, especially in the third trimester. Despite the increased relapse risk in the postpartum period, there is no indication of an adverse effect of childbirth on the long-term course of MS. Fertility treatment in MS has been associated with an increased relapse risk in the following 3-month period, especially when the procedure did not result in pregnancy and gonadotrophin-releasing hormone agonists were used. Altogether, there is substantial evidence to support a regulatory role of sex steroid hormones in MS. In the absence of correlations with single hormone blood levels, we can only speculate about the underlying mechanisms. In conclusion, the increased MS risk in women and the changes in relapse and progression risk in association with reproductive events suggest significant and complex interactions between immune, neuroendocrine and reproductive systems in MS.

In traditional Kaplan-Meier or Cox regression analysis, usually a risk factor measured at baseline is related to mortality thereafter. During follow-up, however, things may change: either the effect of a fixed baseline risk factor may vary over time, resulting in a weakening or strengthening of associations over time, or the risk factor itself may vary over time. In this paper, short-term versus long-term effects (so-called time-dependent effects) of a fixed baseline risk factor are addressed. An example is presented showing that underweight is a strong risk factor for mortality in dialysis patients, especially in the short run. In contrast, overweight is a risk factor for mortality, which is stronger in the long run than in the short run. In addition, the analysis of how time-varying risk factors (so-called time-dependent risk factors) are related to mortality is demonstrated by paying attention to the pitfall of adjusting for sequelae. The proper analysis of effects over time should be driven by a clear research question. Both kinds of research questions, that is those of time-dependent effects as well those of time-dependent risk factors, can be analyzed with time-dependent Cox regression analysis. It will be shown that using time-dependent risk factors usually implies focusing on short-term effects only.


Question: Peroxynitrite is necessary for the nitration of TCR/CD8.

Modified Antigen Perception: A Route to CD8+ T Cell Tolerance in Tumors

The induction of antigen-specific CD8+ T-cell tolerance by myeloid-derived suppressor cells (MDSCs) is a key mechanism by which tumors evade the immune system. In this study, utilizing in vivo models, we reveal that MDSCs can directly interfere with the binding of specific peptide-major histocompatibility complex (pMHC) dimers to T cells expressing CD8, by targeting tyrosine residues in the T-cell receptor (TCR)-CD8 complex for nitration. This process results in the impairment of CD8-expressing T cells' ability to recognize and react to their specific peptide antigens, while preserving their responsiveness to non-specific stimuli. The nitration of TCR-CD8 is triggered by MDSCs through the overproduction of reactive oxygen species and peroxynitrite, occurring during direct cell-to-cell interactions. Computational modeling indicates potential sites of nitration that could alter the conformational dynamics of TCR-CD8, thereby affecting its binding affinity for pMHC. Our findings uncover a previously unrecognized pathway of T-cell tolerance in cancer, which may also be relevant to a variety of diseases characterized by the accumulation of MDSCs.

T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC-activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC-imprinted T cells protect against influenza more effectively than do gut and skin DC-imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC-activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC-activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4.

Thymic-derived natural T regulatory cells (Tregs) are characterized by functional and phenotypic heterogeneity. Recently, a small fraction of peripheral Tregs has been shown to express Klrg1, but it remains unclear as to what extent Klrg1 defines a unique Treg subset. In this study, we show that Klrg1(+) Tregs represent a terminally differentiated Treg subset derived from Klrg1(-) Tregs. This subset is a recent Ag-responsive and highly activated short-lived Treg population that expresses enhanced levels of Treg suppressive molecules and that preferentially resides within mucosal tissues. The development of Klrg1(+) Tregs also requires extensive IL-2R signaling. This activity represents a distinct function for IL-2, independent from its contribution to Treg homeostasis and competitive fitness. These and other properties are analogous to terminally differentiated short-lived CD8(+) T effector cells. Our findings suggest that an important pathway driving Ag-activated conventional T lymphocytes also operates for Tregs.

The transcription factor Kruppel-like factor 2 (KLF2) was proposed to regulate genes involved in cell cycle entry and T cell trafficking; however, the physiological role of its expression in postactivated T cells is not well defined. Previous studies suggested that the cytokines IL-2 and IL-15 differentially regulate KLF2 re-expression in postactivation T cells and that these cytokines also influence effector versus memory T cell differentiation. Using conditional and inducible KLF2-knockout model systems, we tested the specific role of KLF2 expression in activated CD8(+) T cells cultured with these cytokines. KLF2 was required for effective transcription of sphingosine-1-phosphate receptor-1 (S1P(1)) and CD62L in postactivation T cells. However, although different cytokines dramatically altered the expression of cell-cycle-related genes, endogenous KLF2 had a minimal impact. Correspondingly, KLF2-deficient T cells showed dysregulated trafficking but not altered proliferative characteristics following in vivo responses to Ag. Thus, our data help to define KLF2-dependent and -independent aspects of activated CD8(+) T cell differentiation and argue against a physiological role in cell cycle regulation.

Two studies were performed to investigate the effects of an acute bout of physical exercise on the nuclear protein kappaB (NF-kappaB) signaling pathway in rat skeletal muscle. In Study 1, a group of rats (n=6) was run on the treadmill at 25 m/min, 5% grade, for 1 h or until exhaustion (Ex), and compared with a second group (n=6) injected with two doses of pyrrolidine dithiocarbamate (PDTC, 100 mg/kg, i.p.) 24 and 1 h prior to the acute exercise bout. Three additional groups of rats (n=6) were injected with either 8 mg/kg (i.p.) of lipopolysaccharide (LPS), 1 mmol/kg (i.p.) t-butylhydroperoxide (tBHP), or saline (C) and killed at resting condition. Ex rats showed higher levels of NF-kappaB binding and P50 protein content in muscle nuclear extracts compared with C rats. Cytosolic IkappaBalpha and IkappaB kinase (IKK) contents were decreased, whereas phospho-IkappaBalpha and phospho-IKK contents were increased, comparing Ex vs. C. The exercise-induced activation of NF-kappaB signaling cascade was partially abolished by PDTC treatment. LPS, but not tBHP, treatment mimicked and exaggerated the effects observed in Ex rats. In Study 2, the time course of exercise-induced NF-kappaB activation was examined. Highest levels of NF-kappaB binding were observed at 2 h postexercise. Decreased cytosolic IkappaBalpha and increased phosphor-IkappaBalpha content were found 0-1 h postexercise whereas P65 reached peak levels at 2-4 h. These data suggest that the NF-kappaB signaling pathway can be activated in a redox-sensitive manner during muscular contraction, presumably due to increased oxidant production. The cascade of intracellular events may be the overture to elevated gene expression of manganese superoxide dismutase reported earlier (Pfluegers Arch. 442, 426-434, 2001).

Metastatic melanoma is a rapidly progressing disease with high mortality rate and limited treatment options. Immunotherapy based on tumor-targeting cytotoxic T cell responses represents a promising strategy. To assist in its development, we examined the possibility and efficacy of using CD4+ cytotoxic T cells. The regulatory mechanisms controlling CD4+ T cell-mediated cytotoxicity were also investigated. We found that naturally occurring granzyme B and perforin-expressing CD4+ cytotoxic T cells can be recovered from metastatic melanoma patients at significantly elevated frequencies compared to those from healthy controls. These CD4+ cytotoxic T cells were also capable of killing autologous tumor cells harvested from metastatic melanoma, independent of CD8+ T cells or any other cell types. However, several restricting factors were observed. First, the cytolytic activity by CD4+ T cells required high MHC class II expression on melanoma cells, which was not satisfied in a subset of melanomas. Second, the granzyme B and perforin release by activated CD4+ cytotoxic T cells was reduced after coculturing with autologous melanoma cells, characterized by low LAMP-1 expression and low granzyme B and perforin secretion in the supernatant. This suggested that inhibitory mechanisms were present to suppress CD4+ cytotoxic T cells. Indeed, blockade of PD-1 and CTLA-4 had increased the cytolytic activity of CD4+ T cells but was only effective in MHC class II high but not MHC class II low melanomas. Together, our study showed that CD4+ T cell-mediated cytotoxicity could eliminate primary melanoma cells but the efficacy depended on MHC class II expression.

Patients with essential hypertension exhibit blunted endothelium-dependent vasodilator responses, which may be largely attributable to reduced bioactivity of nitric oxide (NO). Therefore, we measured the end product of NO, nitrate plus nitrite (nitrogen oxide), and examined the relationship between the degree of hypertension and plasma nitrate plus nitrite levels in patients with essential hypertension. The combined plasma concentration of nitrate plus nitrite, end products of NO metabolism, was reduced in individuals with essential hypertension relative to that in control subjects (15.7+/-1.1 versus 22.8+/-1.4 mmol x L(-1), P<.001); individuals with borderline hypertension showed values that were intermediate between those of the other two groups (18.2+/-1.2 mmol x L(-1), P<.001). The plasma nitrogen oxide concentration showed significant inverse correlations with both systolic and diastolic blood pressures. The basal concentration of nitrogen oxide in the plasma was reduced, at least in the peripheral circulation, in individuals with essential hypertension.

NADPH oxidase is a critical regulator of both antimicrobial host defense and inflammation. Activated in nature by microbes and microbial-derived products, the phagocyte NADPH oxidase is rapidly assembled, and generates reactive oxidant intermediates (ROIs) in response to infectious threat. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent and severe bacterial and fungal infections, and pathology related to excessive inflammation. Studies in CGD patients and CGD mouse models indicate that NADPH oxidase plays a key role in modulating inflammation and injury that is distinct from its antimicrobial function. The mechanisms by which NADPH oxidase mediates killing of pathogens and regulation of inflammation have broad relevance to our understanding of normal physiological immune responses and pathological states, such as acute lung injury and bacterial or fungal infections.

Regulatory T (Treg) cells, whose differentiation and function are controlled by X chromosome-encoded transcription factor Foxp3, are generated in the thymus (tTreg) and extrathymically (peripheral, pTreg), and their deficiency results in fatal autoimmunity. Here, we demonstrate that a Foxp3 enhancer, conserved noncoding sequence 1 (CNS1), essential for pTreg but dispensable for tTreg cell generation, is present only in placental mammals. CNS1 is largely composed of mammalian-wide interspersed repeats (MIR) that have undergone retrotransposition during early mammalian radiation. During pregnancy, pTreg cells specific to a model paternal alloantigen were generated in a CNS1-dependent manner and accumulated in the placenta. Furthermore, when mated with allogeneic, but not syngeneic, males, CNS1-deficient females showed increased fetal resorption accompanied by increased immune cell infiltration and defective remodeling of spiral arteries. Our results suggest that, during evolution, a CNS1-dependent mechanism of extrathymic differentiation of Treg cells emerged in placental animals to enforce maternal-fetal tolerance.

Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.

Because arginase hydrolyzes arginine to produce ornithine and urea, it has the potential to regulate nitric oxide (NO) and polyamine synthesis. We tested whether expression of the cytosolic isoform of arginase (arginase I) was limiting for NO or polyamine production by activated RAW 264.7 macrophage cells. RAW 264.7 cells, stably transfected to overexpress arginase I or beta-galactosidase, were treated with interferon-gamma to induce type 2 NO synthase or with lipopolysaccharide or 8-bromo-cAMP (8-BrcAMP) to induce ornithine decarboxylase. Overexpression of arginase I had no effect on NO synthesis. In contrast, cells overexpressing arginase I produced twice as much putrescine after activation than did cells expressing beta-galactosidase. Cells overexpressing arginase I also produced more spermidine after treatment with 8-BrcAMP than did cells expressing beta-galactosidase. Thus endogenous levels of arginase I are limiting for polyamine synthesis, but not for NO synthesis, by activated macrophage cells. This study also demonstrates that it is possible to alter arginase I levels sufficiently to affect polyamine synthesis without affecting induced NO synthesis.


Question: Pleiotropic linking of GLP-1R to cellular effectors inside the cell advances unique patterns of signal transduction.

The Molecular Basis of Biased Agonism at the GLP-1 Receptor: A Key to Tailored Therapeutics

Ligand-directed signal bias at G protein-coupled receptors (GPCRs) presents a promising avenue for therapeutic development, offering the potential for more effective and safer drugs by selectively modulating cellular signaling pathways. A fundamental aspect of leveraging signal bias is elucidating the molecular mechanisms that connect ligand binding to downstream intracellular responses. Class B GPCRs, including the glucagon-like peptide-1 receptor (GLP-1R), are activated through the interaction of the N-terminal region of their peptide ligands with the receptor core. To gain insights into the molecular events underlying signal transduction, we employed advanced analytical techniques to distinguish between effects on pathway-specific signaling and modifications to agonist affinity. By mapping these effects onto three-dimensional models of the receptor-ligand complex, we obtained detailed molecular insights into the initial stages of receptor activation and the mechanisms underlying biased agonism. Our findings demonstrate that peptide agonists can interact with distinct regions of the receptor's extracellular surface to achieve selective effector coupling and biased signaling, laying the groundwork for the rational design of biased agonists that can finely tune cellular responses.

Fibroblast growth factor 21 (FGF21) is a recently discovered metabolic regulator. Exogenous FGF21 produces beneficial metabolic effects in animal models; however, the translation of these observations to humans has not been tested. Here, we studied the effects of LY2405319 (LY), a variant of FGF21, in a randomized, placebo-controlled, double-blind proof-of-concept trial in patients with obesity and type 2 diabetes. Patients received placebo or 3, 10, or 20 mg of LY daily for 28 days. LY treatment produced significant improvements in dyslipidemia, including decreases in low-density lipoprotein cholesterol and triglycerides and increases in high-density lipoprotein cholesterol and a shift to a potentially less atherogenic apolipoprotein concentration profile. Favorable effects on body weight, fasting insulin, and adiponectin were also detected. However, only a trend toward glucose lowering was observed. These results indicate that FGF21 is bioactive in humans and suggest that FGF21-based therapies may be effective for the treatment of selected metabolic disorders.

The ideal anti-obesity drug would produce sustained weight loss with minimal side effects. The mechanisms that regulate energy balance have substantial built-in redundancy, overlap considerably with other physiological functions, and are influenced by social, hedonic and psychological factors that limit the effectiveness of pharmacological interventions. It is therefore unsurprising that anti-obesity drug discovery programmes have been littered with false starts, failures in clinical development, and withdrawals due to adverse effects that were not fully appreciated at the time of launch. Drugs that target pathways in metabolic tissues, such as adipocytes, liver and skeletal muscle, have shown potential in preclinical studies but none has yet reached clinical development. Recent improvements in the understanding of peptidergic signalling of hunger and satiety from the gastrointestinal tract mediated by ghrelin, cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), and of homeostatic mechanisms related to leptin and its upstream pathways in the hypothalamus, have opened up new possibilities. Although some have now reached clinical development, it is uncertain whether they will meet the strict regulatory hurdles required for licensing of an anti-obesity drug. However, GLP-1 receptor agonists have already succeeded in diabetes treatment and, owing to their attractive body-weight-lowering effects in humans, will perhaps also pave the way for other anti-obesity agents. To succeed in developing drugs that control body weight to the extent seen following surgical intervention, it seems obvious that a new paradigm is needed. In other therapeutic arenas, such as diabetes and hypertension, lower doses of multiple agents targeting different pathways often yield better results than strategies that modify one pathway alone. Some combination approaches using peptides and small molecules have now reached clinical trials, although recent regulatory experience suggests that large challenges lie ahead. In future, this polytherapeutic strategy could possibly rival surgery in terms of efficacy, safety and sustainability of weight loss.

Mammalian cells depend on growth factor signaling to take up nutrients; however, coordination of glucose and glutamine uptake has been a mystery. In this issue of Genes & Development, Wellen and colleagues (pp. 2784-2799) show that glucose flux through the hexosamine biosynthesis pathway regulates growth factor receptor glycosylation and enables glutamine consumption. This mechanism ensures that cells do not engage in anabolic metabolism when nutrients are limiting, and highlights how substrate availability for protein modifications can modulate cell signaling.

The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) and that both pathways are modulated by their respective endogenous receptor ligands. By addition of specific pathway modulators against the G protein subunit Galphai, phospholipase C, protein kinase C, calcineurin, p38 MAP kinase, and MEK1, we find that the constitutive and ligand-dependent inductions are mediated by multiple yet similar pathways in both receptors. The NFAT and CREB transcription factors and their upstream activators are known inducers of host and virally encoded genes. We propose that the activity of these virally encoded chemokine receptors coordinates host and potentially viral gene expression similarly. As ORF74 is a known inducer of neoplasia, these findings may have important implications for cytomegalovirus-associated pathogenicity.

How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.

Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.

The SNF1/AMP-activated protein kinase (AMPK) family maintains the balance between ATP production and consumption in all eukaryotic cells. The kinases are heterotrimers that comprise a catalytic subunit and regulatory subunits that sense cellular energy levels. When energy status is compromised, the system activates catabolic pathways and switches off protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation. Surprisingly, recent results indicate that the AMPK system is also important in functions that go beyond the regulation of energy homeostasis, such as the maintenance of cell polarity in epithelial cells.

Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa.

The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.

In this study, we present data showing that two members of the GRAS family of genes from rice, CIGR1 and CIGR2(chitin-inducible gibberellin-responsive), inducible by the potent elicitor N-acetylchitooligosaccharide (GN), are rapidly induced by exogenous gibberellins. The pattern of mRNA accumulation was dependent on the dose and biological activity of the gibberellins, suggesting that the induction of the genes by gibberellin is mediated by a biological receptor capable of specific recognition and signal transduction upon perception of the phytoactive compounds. Further pharmacological analysis revealed that the CIGR1 and CIGR2 mRNA accumulation by treatment with gibberellin is dependent upon protein phosphorylation/dephosphorylation events. In rice calli derived from slender rice 1, a constitutive gibberellin-responsive mutant, or d1, a mutant deficient in the α-subunit of the heterotrimeric G-protein, CIGR1 and CIGR2 were induced by a GN elicitor, yet not by gibberellin. Neither gibberellin nor GN showed related activities in defense or development, respectively. These results strongly suggested that the signal transduction cascade from gibberellin is independent of that from GN, and further implied that CIGR1 and CIGR2 have dual, distinct roles in defense and development.