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To examine the role of adipose-resident macrophages in insulin resistance, we examined the gene expression of CD68, a macrophage marker, along with macrophage chemoattractant protein-1 (MCP-1) in human subcutaneous adipose tissue using real-time RT-PCR. Both CD68 and MCP-1 mRNAs were expressed in human adipose tissue, primarily in the stromal vascular fraction. When measured in the adipose tissue from subjects with normal glucose tolerance, covering a wide range of BMI (21-51 kg/[m.sup.2]) and insulin sensitivity ([S.sub.I]) (0.6-8.0 x [10.sup.-4] [min.sup.-1] x µ[U.sup.-1] x [ml.sup.-1]), CD68 mRNA abundance, which correlated with the number of CD68-positive cells by immunohistochemistry, tended to increase with BMI but was not statistically significant. However, there was a significant inverse relation between CD68 mRNA and SI (r = -0.55, P = 0.02). In addition, there was a strong positive relationship among adipose tissue CD68 mRNA, tumor necrosis factor-a (TNF-α) secretion in vitro (r = 0.79, P < 0.005), and plasma interleukin-6 (r = 0.67, P < 0.005). To determine whether improving [S.sub.I] in subjects with impaired glucose tolerance (IGT) was associated with decreased CD68 expression, IGT subjects were treated for 10 weeks with pioglitazone or metformin. Pioglitazone increased [S.sub.I] by 60% and in the same subjects reduced both CD68 and MCP-1 mRNAs by >50%. Furthermore, pioglitazone resulted in a reduction in the number of CD68-positive cells in adipose tissue and reduced plasma TNF-α. Metformin had no effect on any of these measures. Thus, treatment with pioglitazone reduces expression of CD68 and MCP-1 in adipose tissue, apparently by reducing macrophage numbers, resulting in reduced inflammatory cytokine production and improvement in [S.sub.I]., The increasing prevalence of obesity over the last few decades has been associated with a parallel increase in diabetes and metabolic syndrome (1-3). Although there is a significant correlation between [...]

Metabolic syndrome and type 2 diabetes mellitus are associated with an increased number of macrophage cells that infiltrate white adipose tissue (WAT). Previously, we demonstrated that the treatment of subjects with impaired glucose tolerance (IGT) with the peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone resulted in a decrease in macrophage number in adipose tissue. Here, adipose tissue samples from IGT subjects treated with pioglitazone were examined for apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL-positive cells were identified, and there was a significant 42% increase in TUNEL-positive cells following pioglitazone treatment. Overlay experiments with anti-CD68 antibody demonstrated that most of the TUNEL-positive cells were macrophages. To determine whether macrophage apoptosis was a direct or indirect effect of pioglitazone treatment, human THP1 cells were treated with pioglitazone in vitro, demonstrating increased TUNEL staining in a dose- and time-dependent manner. Furthermore, the appearance of the active proteolytic subunits of caspase-3 and caspase-9 were detected in cell lysate from THP1 cells and also increased in a dose- and time-dependent manner following pioglitazone treatment. Pretreatment with a PPARγ inhibitor, GW9662, prevented pioglitazone induction of the apoptotic pathway in THP1 cells. Differentiated human adipocytes did not show any significant increase in apoptosis after treatment in vitro with piolgitazone. These findings indicate that PPARγ has distinct functions in different cell types in WAT, such that pioglitazone reduces macrophage infiltration by inducing apoptotic cell death specifically in macrophages through PPARγ activation.

Adult myoblasts retain plasticity in developmental potential and can be induced to undergo myogenic, adipogenic, or osteoblastogenic differentiation in vitro. In this report, we show that the balance between myogenic and adipogenic potential in myoblasts is controlled by Wnt signaling. Furthermore, this balance is altered during aging such that aspects of both differentiation programs are coexpressed in myoblasts due to decreased Wnt10b abundance. Mimicking Wnt signaling in aged myoblasts through inhibition of glycogen synthase kinase or through overexpression of Wnt10b resulted in inhibition of adipogenic gene expression and sustained or enhanced myogenic differentiation. On the other hand, myoblasts isolated from Wnt10b null mice showed increased adipogenic potential, likely contributing to excessive lipid accumulation in actively regenerating myofibers in vivo in Wnt10b-/- mice. Whereas Wnt10b deficiency contributed to increased adipogenic potential in myoblasts, the augmented myogenic differentiation potential observed is likely the result of a compensatory increase in Wnt7b during differentiation of Wnt10b-/- myoblasts. No such compensation was apparent in aged myoblasts and in fact, both Wnt5b and Wnt10b were down-regulated. Thus, alteration in Wnt signaling in myoblasts with age may contribute to impaired muscle regenerative capacity and to increased muscle adiposity, both characteristic of aged muscle.

Adult myoblasts retain plasticity in developmental potential and can be induced to undergo myogenic, adipogenic, or osteoblastogenic differentiation in vitro. In this report, we show that the balance between myogenic and adipogenic potential in myoblasts is controlled by Wnt signaling. Furthermore, this balance is altered during aging such that aspects of both differentiation programs are coexpressed in myoblasts due to decreased Wnt10b abundance. Mimicking Wnt signaling in aged myoblasts through inhibition of glycogen synthase kinase or through overexpression of Wnt10b resulted in inhibition of adipogenic gene expression and sustained or enhanced myogenic differentiation. On the other hand, myoblasts isolated from Wnt10b null mice showed increased adipogenic potential, likely contributing to excessive lipid accumulation in actively regenerating myofibers in vivo in Wnt10b-/- mice. Whereas Wnt10b deficiency contributed to increased adipogenic potential in myoblasts, the augmented myogenic differentiation potential observed is likely the result of a compensatory increase in Wnt7b during differentiation of Wnt10b-/- myoblasts. No such compensation was apparent in aged myoblasts and in fact, both Wnt5b and Wnt10b were down-regulated. Thus, alteration in Wnt signaling in myoblasts with age may contribute to impaired muscle regenerative capacity and to increased muscle adiposity, both characteristic of aged muscle.

Advances in quantitative analysis and molecular genotyping have provided unprecedented opportunities to add biological sampling and genetic information to epidemiologic studies. The purpose of this article is to describe the incorporation of DNA sample collection into the National Birth Defects Prevention Study (NBDPS), an ongoing case-control study in an eight-state consortium with a primary goal to identify risk factors for birth defects. Babies with birth defects are identified through birth defects surveillance systems in the eight participating centers. Cases are infants with one or more of over 30 major birth defects. Controls are infants without defects from the same geographic area. Epidemiologic information is collected through an hour-long interview with mothers of both cases and controls. We added the collection of buccal cytobrush DNA samples for case-infants, control-infants, and their parents to this study. We describe here the methods by which the samples have been collected and processed, establishment of a centralized resource for DNA banking, and quality control, database management, access, informed consent, and confidentiality issues. Biological sampling and genetic analyses are important components to epidemiologic studies of birth defects aimed at identifying risk factors. The DNA specimens collected in this study can be used for detection of mutations, study of polymorphic variants that confer differential susceptibility to teratogens, and examination of interactions among genetic risk factors. Information on the methods used and issues faced by the NBDPS may be of value to others considering the addition of DNA sampling to epidemiologic studies. Teratology 3:177–184, 2002. Published 2002 Wiley-Liss, Inc.

The murine 3T3-L1 preadipocyte cell line is well characterized for its capacity to undergo differentiation into adipocytes under appropriate hormonal stimulation. p107, a member of the retinoblastoma tumor suppressor gene family has been shown to be dramatically upregulated during the early requisite clonal expansion phase of 3T3-L1 adipogenesis; however, a functional consequence has yet to be described. A phosphorothioate antisense RNA approach was utilized to determine if inhibition of p107 expression would block or perturb adipocyte differentiation. A series of three phosphorothioate oligonucleotides in antisense orientation was generated, designated AS1, AS2, and AS3 along with a sense control oligonucleotide complementary to AS1 and added to postconfluent cells at a concentration of 20 and 50 μM throughout hormonally stimulated differentiation. Treatment of cells with either concentration of the sense, AS1, AS2, or 20 μM AS3 oligonucleotides had little effect on either Oil Red O lipid accumulation or induction of p107 protein levels. In contrast, treatment with 50 μM AS3 inhibited the increase in p107 protein levels and led to a complete block in differentiation as detected by Oil Red O lipid accumulation and inhibition of adipocyte-specific mRNA expression. In addition, treatment with AS3 led to a significant inhibition of cellular proliferation associated with clonal expansion. Combined, these results provide strong evidence supporting a functional role for p107 in 3T3-L1 adipocyte differentiation.

Continuous infusion of ethanol-containing diets has been demonstrated to generate well-defined pulses in blood and urine ethanol concentrations that occur with a frequency of approximately 6 days. The present study aimed to determine if hepatic class I alcohol dehydrogenase was the cause of these cycles. Adult male rats were fed an ethanol-containing diet by continuous intragastric infusion. Hepatic ADH activity, class I ADH mRNA level and rate of class I ADH gene transcription fluctuated in a cyclic pattern that positively correlated with UECs, and inhibition of ADH with 4-methylpyrazole abolished the UEC pulses. These data demonstrate for the first time an ethanol-dependent regulation of rat hepatic class I ADH. The cyclic behavior of the ethanol levels correlates with changes in class I ADH expression and implies adaptability of the ethanol eliminating system to high concentrations of alcohol.

The inhibitory effects of TNFα on adipocyte differentiation are well described, however, the mechanisms are poorly understood. Early during hormonally-induced 3T3-L1 preadipocyte differentiation there is a requisite mitotic clonal expansion phase that is associated with significant regulation in p130 and p107 protein levels, two members of the retinoblastoma protein family that regulate cell cycle events through interactions with the E2F transcription factors. This regulation occurs within the first 24 hours of differentiation (Day 1) and is characterized by a transient increase in p107 protein and mRNA levels as well as a transient decrease in p130 protein levels. Here we describe that TNFα disrupts the normal pattern of expression of both p130 and p107 proteins, leading to a complete block in mitotic clonal expansion. Interestingly, TNFα-treated cells enter S-phase as determined by 5-bromo-2′-deoxyuridine uptake experiments, but rather than completing cell cycle, they are stimulated to undergo apoptosis.

The homeobox family of proteins are transcription factors are known to be important during the differentiation of a variety of mammalian tissues, however, expression of the genes encoding homeobox proteins during adipogenesis or in adipose tissue has not been described. To investigate whether members of the homeobox gene family are expressed and regulated during adipocyte differentiation, RNA was isolated from 3T3-L1 preadipocyte cells during the hormonal induced differentiation of this cell line into adipocytes. A reverse transcriptase-polymerase chain reaction strategy using degenerate oligonucleotide primers complementary to the highly conserved homeodomain resulted in the identification of 10 different homeobox genes expressed during 3T3-L1 adipogenesis. One of the clones appears to be unique and 9 of the clones represented known members of the homeobox gene family. Examination of the relative mRNA levels encoding these proteins by ribonuclease protection assay during adipocyte differentiation revealed that 3 members, Hox a4, Hox a7, and Hox d4, are regulated as a function of adipocyte development. Further examination of RNA isolated from murine retroperitoneal adipose tissue revealed that these three regulated homeobox mRNAs are expressedin vivo.Combined, these results suggest that members of the homeobox gene family may serve an important role during the differentiation of adipocytes.

Probing total cellular and poly [A+] RNA isolated from various rat tissues with a full-length cDNA encoding the hamster α1-adrenergic receptor results in detection of two transcripts, 3.3 kb and 2.7 kb, which probably both encode the α1b-adrenergic receptor subtype. Both the 3.3 kb and 2.7 kb mRNAs were found to be associated with hepatic polysomes which suggests that these mRNA species are translated into protein. Using non-overlaping 5' and 3' cDNA probes, large sequence differences were not evident between the 3.3 kb and 2.7 kb mRNAs, although the 3'-probe hybridized to a 4.0 kb mRNA in addition to the two smaller transcripts in poly [A+] RNA isolated from renal cortex, but not other tissues. The relative amounts of the 3.3 kb and 2.7 kb mRNAs varied considerably among the five tissues studied. However, the ratio of the two transcripts remained relatively constant in the same tissue taken from animals at different developmental ages. Currently, the physiological significance of multiple α1b-adrenergic receptor gene transcripts is unclear. However, our results suggest that α1b-adrenergic receptor gene expression in the rat is under complex regulatory control that in part is tissue-dependent.

The Differentiation-Specific Element Binding Protein (DSEB) was identified by binding to a specific cis-acting DNA element (DSE) responsible for the irreversible continued expression of the angiotensinogen gene after differentiation of 3T3-L1 adipoblasts to adipocytes. It was also identified as the large subunit of the Replication Factor C complex. During 3T3-L1 adipoblast differentiation, DSEB is induced early and interacts with the DSE that is essential for the sustained transcriptional activation of the angiotensinogen gene. Here we describe loss of function studies in 3T3-L1 cells performed with antisense phosphorothioate oligonucleotides that hybridize to DSEB mRNA. Treatment with 15, 25, and 50 μM antisense DSEB resulted in a dose-dependent inhibition of differentiation-specific lipid accumulation after 3 days of hormonal stimulation. Similar treatment also markedly reduced differentiation-dependent expression of mRNAs encoding angiotensinogen and the fat-specific fatty acid binding protein, aP2. Further, 50 μM antisense DSEB treatment resulted in a significant ∼50% inhibition of the cell proliferation that occurs early in 3T3-L1 adipogenesis. Control experiments using the DSEB sense oligonucleotide had no effect on hormonal-stimulated adipocyte differentiation. Combined, these results suggest that DSEB serves an important role during the proliferative phase of 3T3-L1 adipoblast differentiation.

The potential of infant diet to influence fat cell development has largely been examined in clinical studies with conflicting results. In this study, the direct effects of two standard infant formulas, Enfamil and Similac, as well as human milk were examined using a well characterized model of adipocyte differentiation, the 3T3-L1 murine preadipocyte cell line. After exposure to a hormonal regimen of insulin, dexamethasone, and 1-methyl-3-isobutylmethylxanthine, these cells undergo a mitotic expansion phase followed by terminal differentiation. On d 4 of hormonal exposure, greater than 95% of 3T3-L1 cells exhibit the morphologic and biochemical characteristics of mature adipocytes. In this study, cells were exposed to control medium, or control medium supplemented with either 10% Enfamil, 10% Similac, 10% human milk (skim or whole), or the standard hormonal regimen. Oil Red O-detectable lipid accumulation, immunocytochemical cell proliferation assays, and activated expression of adipocyte differentiation-specific mRNAs by Northern blot analysis were used to assess the effects of treatment on adipocyte differentiation. Results from each level of assessment revealed that both Enfamil and human milk were as effective as the standard hormonal regimen at stimulating adipocyte differentiation. In contrast, results from treatment with Similac or human skim milk were indistinguishable from control unstimulated cells. This study, demonstrating that Enfamil and human milk are capable of independently inducing in vitro adipocyte differentiation, suggests that diet during infancy could influence body fat development.

We present a testable mechanism of low-scale baryogenesis and dark matter production in which neither the baryon nor lepton number are violated. Charged D mesons are produced out of equilibrium at tens of MeV temperatures. The D mesons quickly undergo CP-violating decays to charged pions, which then decay into dark-sector leptons without violating the lepton number. To transfer this lepton asymmetry to the baryon asymmetry, the dark leptons scatter on additional dark-sector states charged under the lepton and baryon number. Amusingly, this transfer proceeds without electroweak sphalerons, which are no longer active at such low scales. We present two example models which can achieve this transfer while remaining consistent with current limits. The required amount of CP violation in charged D meson decays, while currently allowed, will be probed by colliders. Additionally, the relevant decays of charged pions to dark-sector leptons have been constrained by the PIENU and Paul Scherrer Institute experiments and will be further explored in upcoming experiments. [ABSTRACT FROM AUTHOR]

The mirror twin Higgs (MTH) addresses the little hierarchy problem by relating every Standard Model (SM) particle to a twin copy, but is in tension with cosmological bounds on light degrees of freedom. Asymmetric reheating has recently been proposed as a simple way to fix MTH cosmology by diluting the twin energy density. We show that this dilution sets the stage for an interesting freeze-in scenario where both the initial absence of dark sector energy and the feeble coupling to the SM are motivated for reasons unrelated to dark matter production. We give the twin photon a Stueckelberg mass and freeze-in twin electron and positron dark matter through the kinetic mixing portal. The kinetic mixing required to obtain the dark matter abundance is of the loop-suppressed order expected from infrared contributions in the MTH. [ABSTRACT FROM AUTHOR]

A recent cosmological bound on the gravitino mass, m3/2<4.7  eV, together with LHC results on the Higgs mass and direct searches, excludes minimal gauge mediation with high reheating temperatures. We discuss a minimal, vector-mediated model which incorporates the seesaw mechanism for neutrino masses, allows for thermal leptogenesis, ameliorates the μ problem, and achieves the observed Higgs mass and a gravitino as light as 1-2 eV. [ABSTRACT FROM AUTHOR]

Two kinds of fat tissue are present in mammals: white adipose fat (WAT) and brown adipose fat (BAT). Exposure to a cold environment in the winter activates BAT, which defends against the cold by releasing energy as heat. Increased energy expenditure associated with BAT activation has been shown to prevent obesity in rodents. There is an inverse relationship between activation of BAT and adiposity, and elevated inflammation is associated with increased WAT depots. In addition to BAT activation, the thermogenic capacity of rodents can be increased by browning, a process in which some WAT depots are converted to beige fat (mixture of brown and white fat). As with BAT activation, browning appears to be a defense against cold and obesity. Both browning and BAT activation increase expression of a mitochondrial protein called UCP1 that uncouples oxidative respiration to generate heat. Another protein called PGC1 appears to act synergistically with UDP1 to activate brown fat in a cold environment. Preliminary data suggest that brown fat may also be a defense against cold and obesity in humans, but it is unclear whether adult humans exposed to the cold have the ability to transform some white fat deposits into beige fat.

We study a new class of signals where fermionic dark matter is absorbed by bound electron targets. Fermionic absorption signals in direct detection and neutrino experiments are sensitive to dark matter with sub-MeV mass, probing a region of parameter space in which dark matter is otherwise challenging to detect. We calculate the rate and energy deposition spectrum in xenon-based detectors, making projections for current and future experiments. We present two possible models that display fermionic absorption by electrons and study the detection prospects in light of other constraints. [ABSTRACT FROM AUTHOR]

Dark matter could be a thermal relic comprised of strongly interacting massive particles (SIMPs), where 3→2 interactions set the relic abundance. Such interactions generically arise in theories of chiral symmetry breaking via the Wess-Zumino-Witten term. In this work, we show that an axionlike particle can successfully maintain kinetic equilibrium between the dark matter and the visible sector, allowing the requisite entropy transfer that is crucial for SIMPs to be a cold dark matter candidate. Constraints on this scenario arise from beam dump and collider experiments, from the cosmic microwave background, and from supernovae. We find a viable parameter space when the axionlike particle is close in mass to the SIMP dark matter, with strong-scale masses of order a few hundred MeV. Many planned experiments are set to probe the parameter space in the near future. [ABSTRACT FROM AUTHOR]