Impaired atrial natriuretic peptide-mediated lipolysis in obesity.

BACKGROUND: Catecholamines and natriuretic peptides (NPs) are the only hormones with pronounced lipolytic effects in human white adipose tissue. Although catecholamine-induced lipolysis is well known to be impaired in obesity and insulin resistance, it is not known whether the effect of NPs is also altered. METHODS: Catecholamine- and atrial NP (ANP)-induced lipolysis was investigated in abdominal subcutaneous adipocytes in vitro and in situ by microdialysis. RESULTS: In a cohort of 122 women, both catecholamine- and ANP-induced lipolysis in vitro was markedly attenuated in obesity (n=87), but normalized after substantial body weight loss (n=52). The impairment of lipolysis differed between the two hormones when expressing lipolysis per lipid weight, the ratio of stimulated over basal (spontaneous) lipolysis rate or per number of adipocytes. Thus, while the response to catecholamines was lower when expressed as the former two measures, it was higher when expressed per cell number, a consequence of the significantly larger fat cell size in obesity. In contrast, although ANP-induced lipolysis was also attenuated when expressed per lipid weight or the ratio stimulated/basal, it was similar between non-obese and obese subjects when expressed per cell number suggesting that the lipolytic effect of ANP may be even more sensitive to the effects of obesity than catecholamines. Obesity was characterized by a decrease in the protein expression of the signaling NP A receptor (NPRA) and a trend toward increased levels of the clearance receptor NPRC. The impairment in ANP-induced lipolysis observed in vitro was corroborated by microdialysis experiments in situ in a smaller cohort of lean and overweight men. CONCLUSIONS: ANP- and catecholamine-induced lipolysis is reversibly attenuated in obesity. The pro-lipolytic effects of ANP are relatively more impaired compared with that of catecholamines, which may in part be due to specific changes in NP receptor expression.

Adipogenic progenitors from obese human skeletal muscle give rise to functional white adipocytes that contribute to insulin resistance.

BACKGROUND/OBJECTIVES: Recent reports indicate that inter/intramuscular adipose tissue (IMAT), composed by adipocytes underneath the deep fascia of the muscles, is positively correlated with aging, obesity and insulin resistance in humans. However, no molecular/cellular evidence is available to support these interactions. The current study aimed to better characterize human skeletal muscle-derived adipogenic progenitors obtained from obese volunteers and investigate the impact of derived adipocytes on insulin action in primary skeletal muscle cells. METHODS: Primary cultured stroma-vascular fraction (SVF) obtained from vastus lateralis muscle biopsies of middle-aged obese subjects was immunoseparated (magnetic beads or flow cytometry). The characteristics and/or metabolic phenotype of CD56(+), CD56(-) and CD56(-)CD15(+) cellular fractions were investigated by complementary approaches (flow cytometry, cytology, quantitative PCR and metabolic assays). The effects of conditioned media from CD56(-)CD15(+) cells differentiated into adipocytes on insulin action and signaling in human primary myotubes was also examined. RESULTS: Our data indicate that CD56(+) and CD56(-) cellular fractions isolated from cultured SVF of human muscle contain two distinct committed progenitors: CD56(+) cells (that is, satellite cells) as myogenic progenitors and CD15(+) cells as adipogenic progenitors, respectively. CD56(-)CD15(+)-derived adipocytes display the phenotype and metabolic properties of white adipocytes. Secretions of CD56(-)CD15(+) cells differentiated into functional white adipocytes reduced insulin-mediated non-oxidative glucose disposal (P=0.0002) and insulin signaling. CONCLUSIONS: Using in-vitro models, we show for the first time that secretions of skeletal muscle adipocytes are able to impair insulin action and signaling of muscle fibers. This paracrine effect could explain, at least in part, the negative association between high levels of IMAT and insulin sensitivity in obesity and aging.

Distinct phosphoisoforms of the Xenopus Mcm4 protein regulate the function of the Mcm complex.

Initiation of DNA replication in eukaryotes requires the assembly of prereplication complexes (pre-Rcs) at the origins of replication. The assembly and function of the pre-Rcs appear to be controlled by phosphorylation events. In this study we report the detailed characterization of the cell cycle phosphorylation of one component of the Xenopus pre-Rcs, the Mcm protein complex. We show that individual Mcm subunits are differentially phosphorylated during the cell cycle. During mitosis, the Mcm4 subunit is hyperphosphorylated, while the other subunits are not actively phosphorylated. The mitotic phosphorylation of Mcm4 requires Cdc2-cyclin B and other unknown kinases. Following exit from mitosis, the Mcm4 subunit of the cytosolic interphase complex undergoes dephosphorylation, and the Mcm2, Mcm3, or Mcm6 subunits are then actively phosphorylated by kinase(s) other than cyclin-dependent kinases (Cdks) or Cdc7. The association of the Mcm complex with the pre-Rcs correlates with the formation of a transient interphase complex. This complex contains an intermediately phosphorylated Mcm4 subunit and is produced by partial dephosphorylation of the mitotic hyperphosphorylated Mcm4 protein. Complete dephosphorylation of the Mcm4 subunit inactivates the Mcm complex and prevents its binding to the chromatin. Once the Mcm complex is assembled on the chromatin the Mcm4 and the Mcm2 proteins are the only subunits phosphorylated during the activation of the pre-Rcs. These chromatin-associated phosphorylations require nuclear transport and are independent of Cdk2-cyclin E. These results suggest that the changes in Mcm4 phosphorylation regulate pre-Rc assembly and the function of the pre-Rcs on the chromatin.

Evidence for different MCM subcomplexes with differential binding to chromatin in Xenopus.

MCM proteins are molecular components of the DNA replication licensing system in Xenopus. These proteins comprise a conserved family made up of six distinct members which have been found to associate in large protein complexes. We have used a combination of biochemical and cytological methods to study the association of soluble and chromatin-bound Xenopus MCM proteins during the cell cycle. In interphase, soluble MCM proteins are found organized in a core salt-resistant subcomplex that includes MCM subunits which are known to have high affinity for histones. The interphasic complex is modified at mitosis and the subunit composition of the resulting mitotic subcomplexes is distinct, indicating that the stability of the MCM complex is under cell cycle control. Moreover, we provide evidence that the binding of MCM proteins to chromatin may occur in sequential steps involving the loading of distinct MCM subunits. Comparative analysis of the chromatin distribution of MCM2, 3, and 4 shows that the binding of MCM4 is distinct from that of MCM2 and 3. Altogether, these data suggest that licensing of chromatin by MCMs occurs in an ordered fashion involving discrete subcomplexes.

[Clinical evaluation: from intermediate to surrogate criteria]. / Evaluation clinique: du critère intermédiaire au critère de substitution.

The development and evaluation of new drugs often rely on surrogacy. An intermediate outcome becomes a surrogate outcome if it fulfils certain criteria, it should be easier to measure compared with the clinical outcome, a statistical relationship should exist between the clinical outcome and the surrogate outcome, a relation should exist allowing prediction of the degree of clinical effect based on the measured effect on the surrogate outcome. Development and authorization of drugs today often rely on so-called surrogate outcomes. Is this use sound? The validity of such outcomes has been reviewed in different therapeutic areas: hypertension, venous thromboembolism, AIDS, osteoporosis, hepatitis C. Based on this review, a pragmatic strategy is proposed which allows for the validation and proper use of surrogate outcomes.

Chromatin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication.

A Xenopus homologue of Schizosaccharomyces pombe cdc21 has been characterized as a new member of the MCM family of proteins. The cdc21 protein exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 binds to decondensing chromatin at the end of mitosis, localizing to numerous foci which form prior to reconstitution of the nuclear membrane. The association of cdc21 with chromatin occurs in membrane-free high speed extracts and is resistant to detergent extraction. The spatial organization of the cdc21 foci resembles that of pre-replication centres though no co-localization with RP-A was observed. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced as the DNA replication forks progress. These subnuclear changes in localization correlate with cell-cycle-regulated changes in phosphorylation. Cdc21 binds to chromatin in an underphosphorylated state, but in early S phase the nuclear localized cdc21 is partially phosphorylated before it is displaced from the chromatin. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor.

[Women in clinical trials]. / Les femmes dans les essais cliniques.

In a workshop held in Giens in September 1994, representatives of drug companies and scientists met to discuss the place of women in clinical trials. They recommend that, in phase II and III studies, men and women should be included in a proportion equivalent to that observed for the condition studied. They also recommend that the impact of the gender on the results should be systematically studied.

[Survey for participation in therapeutic trials in psychiatry]. / Enquête sur la participation aux essais thérapeutiques en psychiatrie.

In collaboration with the Sofres Institute, a two-phase population survey was conducted in 1991. During the first phase, 10,000 households representative of the French population and regularly surveyed by the Sofres Institute answered five questions by mail. The purpose was to identify subgroups regularly receiving psychotropic drugs and followed up by a physician. They were identified according to whether they used any of some 30 neuroleptic and antidepressant drugs, the most frequently prescribed in their class. According to their answers returned by post, the subjects were classified into three groups: regular psychotropic drug users followed by general practitioners, regular psychotropic drug users followed by specialists (these subjects being considered possibly more severely ill), and non users of these drugs, subjects considered as controls. In the second phase, a random sample of these groups received a detailed questionnaire including 14 questions related to clinical trials. In addition the same questionnaire was given to hospitalised patients in three different psychiatric units. Out of total of 958 subjects who received the detailed questionnaire, 712 answered (a 74% response rate). Results indicate that a majority of the subjects (50% to 74% depending on the subgroups) have already heard about the need for clinical trials to test new drugs. However, less than 20% of these subjects are aware of a recent French law protecting patient rights in clinical trials. This awareness appears to be quite insufficient A large number of subjects do not know whether they have been part of a clinical trial in the past.(ABSTRACT TRUNCATED AT 250 WORDS)

Microtubule depolymerization promotes particle and chromosome movement in vitro.

We have developed a system for studying the motions of cellular objects attached to depolymerizing microtubules in vitro. Radial arrays of microtubules were grown from lysed and extracted Tetrahymena cells attached to a glass coverslip that formed the top of a light microscope perfusion chamber. A preparation of chromosomes, which also contained vesicles, was then perfused into the chamber and allowed to bind to the microtubule array. The concentration of tubulin was then reduced by perfusing buffer that lacked both tubulin and nucleotide triphosphates, and the resulting microtubule depolymerization was observed by light microscopy. A fraction of the bound objects detached in the flow and washed away, while others stabilized the microtubules to which they were bound. Some of the particles and chromosomes, however, moved in toward the Tetrahymena ghost as their associated microtubules shortened. The mean speeds for particles and chromosomes were 26 +/- 20 and 15 +/- 12 microns/min, respectively. These motions occurred when nucleotide triphosphate levels were very low, as a result of either dilution or by the action of apyrase. Furthermore, the motions were unaffected by 100 microM sodium orthovanadate, suggesting that these forces are not the result of ATP hydrolysis by a minus end-directed mechanoenzyme. We conclude that microtubule depolymerization provided the free energy for the motions observed. All the objects that we studied in detail moved against a stream of buffer flowing at approximately 100 microns/s, so that the force being developed was at least 10(-7) dynes. This force is large enough to contribute to some forms of motility in living cells.

Cytoplasmic dynein is localized to kinetochores during mitosis.

Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.

Fluorescent microtubules break up under illumination.

We have synthesized three new fluorescent analogues of tubulin, using fluorescein or rhodamine groups attached to N-hydroxy-succinimidyl esters, and have partially characterized the properties of these analogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laboratories. Our results show that all four analogues assemble into microtubules which break up when exposed to light of the wavelengths that excite fluorescence. This sensitivity places severe constraints on the use of these analogues in studies of microtubule dynamics.

Inhibition of actin polymerization by latrunculin A.

Latrunculin A, a toxin purified from the red sea sponge Latrunculia magnifica, was found previously to induce striking reversible changes in the morphology of mammalian cells in culture and to disrupt the organization of their microfilaments. We now provide evidence that latrunculin A affects the polymerization of pure actin in vitro in a manner consistent with the formation of a 1:1 molar complex between latrunculin A and G-actin. The equilibrium dissociation constant (Kd) for the reaction in vitro is about 0.2 microM whereas the effects of the drug on cultured cells are detectable at concentrations in the medium of 0.1-1 microM.

Effects of serum vitamin-D-binding protein on actin in the presence of plasma gelsolin.

The interaction of serum vitamin-D-binding protein (DBP) and plasma gelsolin with actin was studied using fluorescent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-actin or N-pyrenylcarboxyamidomethyl-actin. DBP and gelsolin formed very tight complexes with one or two monomeric actin subunits respectively. Kd values of about 10 nm have been found for both complexes. When DBP and gelsolin were added simultaneously to G-actin no ternary complex was observed and the DBP-actin complex was formed preferentially. In the presence of CaCl2 no transfer of actin occurred from the 1:2 gelsolin:actin molar complex to free DBP while in the presence of EGTA one actin monomer could be transferred. DBP did not affect the severing activity of gelsolin. The effects of a mixture of gelsolin and DBP on F-actin suggest that only individual interactions of these two plasma proteins with actin occurred in solution.

Interaction of plasma gelsolin with ADP-actin.

In the presence of Ca2+, gelsolin forms a very tight, stoichiometric complex with 2 molecules of ADP-G-actin. Removal of free Ca2+ causes the 1:2 complex to dissociate to a 1:1 complex. Gelsolin accelerates the very slow polymerization of ADP-actin, apparently by accelerating the rate of nucleation, but the number concentration of filaments formed is probably less than the gelsolin concentration, indicating that the GA2 complex is not a true nucleus. These results are similar to those obtained for the interaction of gelsolin with ATP-G-actin. Both kinetic and equilibrium measurements demonstrate that the critical concentration of gelsolin-capped ADP-actin filaments (8 microM in 1 mM MgCl2 and 0.2 mM ADP) is the same as for the uncapped filaments, proving that the critical concentration is the same at both ends of the equilibrium polymer in ADP as predicted by theory. The association and dissociation rate constants for the addition of ADP-G-actin at the pointed end of an ADP-F-actin filament are estimated to be 4.6 X 10(4) M-1 s-1 and 0.4 s-1, respectively, about 15-fold lower than the rate constants at the barbed end.

ATP hydrolysis by the gelsolin-actin complex and at the pointed ends of gelsolin-capped filaments.

To obtain kinetic information about the pointed ends of actin filaments, experiments were carried out in the presence of gelsolin which blocks all events at the kinetically dominant barbed ends. The 1:2 gelsolin-actin complex retains 1 mol/mol of actin-bound ATP, but it neither hydrolyzes the ATP nor exchanges it with ATP free in solution at a significant rate. On the other hand, the actin filaments with their barbed ends capped with gelsolin hydrolyze ATP relatively rapidly at steady state, apparently as a result of the continued interaction of ATP-G-actin with the pointed ends of the filaments. ATP hydrolysis during spontaneous polymerization of actin in the presence of relatively high concentrations of gelsolin lags behind filament elongation so that filaments consisting of as much as 50% ATP-actin subunits are transiently formed. Probably for this reason, during polymerization the actin monomer concentration transiently reaches a concentration lower than the final steady-state critical concentration of the pointed end. At steady state, however, there is no evidence for an ATP cap at the pointed ends of gelsolin-capped filaments, which differs from the barbed ends which do have an ATP cap in the absence of gelsolin. As there is no reason presently to think that gelsolin has any effect on events at the pointed ends of filaments, the properties of the pointed ends deduced from these experiments with gelsolin-capped filaments are presumably equally applicable to the pointed ends of filaments in which the barbed ends are free.

Interaction of plasma gelsolin with G-actin and F-actin in the presence and absence of calcium ions.

Plasma gelsolin formed a very tight 1:2 complex with G-actin in the presence of Ca2+, but no interaction between gelsolin and G-actin was detected in the presence of excess EGTA. However, the 1:2 complex dissociated into a 1:1 gelsolin:actin complex and monomeric actin when excess EGTA was added. Plasma gelsolin bound tightly to the barbed ends of actin filaments and also severed filaments in the presence of Ca2+ and bound weakly to the filament barbed end in the presence of EGTA. The 1:2 gelsolin-actin complex bound to the barbed ends of filaments but did not sever them. By blocking the barbed end of filaments with plasma gelsolin, we determined the critical concentration at the pointed end in 1 mM MgCl2 and 0.2 mM ATP to be 4 microM. The dissociation rate constant for ADP-G-actin from the pointed end was estimated to be about 0.4 s-1 and the association rate constant to be about 5 X 10(4) M-1 s-1. Finally, we obtained evidence that plasma gelsolin accelerates but does not bypass the nucleation step and, therefore, that the concentration of gelsolin does not directly determine the concentration of filaments polymerized in its presence. Thus, gelsolin-capped filaments may not provide an absolutely reliable method for determining the rate constant for the association of ATP-G-actin at the pointed ends of filaments, but a reasonable estimate would be 1 X 10(5) M-1 s-1 in 1 mM MgCl2 and 0.2 mM ATP.

Effect of actin filament length and filament number concentration on the actin-activated ATPase activity of Acanthamoeba myosin I.

The actin-activated Mg2+-ATPase activities of phosphorylated Acanthamoeba myosins IA and IB were previously found to have a highly cooperative dependence on myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This behavior is reflected in the requirement for a higher concentration of F-actin for half-maximal activation of the myosin Mg2+-ATPase at low ratios of myosin:actin (noncooperative phase) than at high ratios of myosin:actin (cooperative phase). These phenomena could be explained by a model in which each molecule of the nonfilamentous myosins IA and IB contains two F-actin-binding sites of different affinities with binding of the lower affinity site being required for expression of actin-activated ATPase activity. Thus, enzymatic activity would coincide with cross-linking of actin filaments by myosin. This theoretical model predicts that shortening the actin filaments and increasing their number concentration at constant total F-actin should increase the myosin concentration required to obtain the cooperative increase in activity and should decrease the F-actin concentration required to reach half-maximal activity at low myosin:actin ratios. These predictions have been experimentally confirmed by shortening actin filaments by addition of plasma gelsolin, an F-actin capping/severing protein. In addition, we have found that actin "filaments" as short as the 1:2 gelsolin-actin complex can significantly activate Acanthamoeba myosin I.

Isolation and characterization of human blood platelet gelsolin.

A 90-kDa protein-actin stable complex was purified from blood platelets by a short and efficient procedure giving at the same time actin used in polymerization assays. 90-kDa protein free of actin was prepared from the complex by 8 M ureau treatment and renaturation. By its molecular mass, immunological cross-reactivity with macrophage gelsolin and its effect on G- and F-actin the 90-kDa protein appears as the platelet gelsolin.

The critical concentration of actin in the presence of ATP increases with the number concentration of filaments and approaches the critical concentration of actin.ADP.

F-actin at steady state in the presence of ATP partially depolymerized to a new steady state upon mechanical fragmentation. The increase in critical concentration with the number concentration of filaments has been quantitatively studied. The data can be explained by a model in which the preferred pathway for actin association-dissociation reactions at steady state in the presence of ATP involves binding of G-actin . ATP to filaments, ATP hydrolysis, and dissociation of G-actin . ADP which is then slowly converted to G-actin . ATP. As a consequence of the slow exchange of nucleotide on G-actin, the respective amounts of G-actin . ATP and G-actin . ADP coexisting with F-actin at steady state depend on the filament number concentration. G-actin coexisting with F-actin at zero number concentration of filaments would then consist of G-actin . ATP only, while the critical concentration obtained at infinite number of filaments would be that for G-actin . ADP. Values of 0.35 and 8 microM, respectively, were found for these two extreme critical concentrations for skeletal muscle actin at 20 degrees C, pH 7.8, 0.1 mM CaCl2, 1 mM MgCl2, and 0.2 mM ATP. The same value of 8 microM was directly measured for the critical concentration of G-actin . ADP polymerized in the presence of ADP and absence of ATP, and it was unaffected by fragmentation. These results have important implications for experiments in which critical concentrations are compared under conditions that change the filament number concentrations.