The educational and professional status of clinical embryology and clinical embryologists in Europe.

STUDY QUESTION: What is the recognition of clinical embryology and the current status of clinical embryologists in European countries, regarding educational levels, responsibilities and workload, and need for a formal education in assisted reproductive technology (ART)? SUMMARY ANSWER: It is striking that the profession of clinical embryology, almost 40 years after the introduction of IVF, is still not officially recognized in most European countries. WHAT IS KNOWN ALREADY: Reproductive medicine has developed into a sophisticated multidisciplinary medical branch since the birth of Louise Brown 37 years ago. The European Board & College of Obstetrics and Gynaecology (EBCOG) has recognized reproductive medicine as a subspeciality and has developed a subspeciality training for gynaecologists in collaboration with the European Society for Human Reproduction and Embryology (ESHRE). However, nothing similar exists for the field of clinical embryology or for clinical embryologists. STUDY DESIGN, SIZE, DURATION: A questionnaire about the situation in clinical embryology in the period of 2012-2013 in the respective European country was sent to ESHRE National representatives (basic scientists only) in December 2013. At this time, 28 European countries had at least one basic scientist in the ESHRE Committee of National Representatives. PARTICIPANTS/MATERIALS, SETTING, METHODS: The survey consisted of 46 numeric, dichotomous (yes/no) or descriptive questions. Answers were obtained from 27 out of 28 countries and the data were tabulated. Data about the numbers of 'ESHRE Certified Embryologists' were taken from the ESHRE Steering Committee for Embryologist Certification. MAIN RESULTS AND THE ROLE OF CHANCE: In 2012, more than 7000 laboratory staff from 1349 IVF clinics in 27 European countries performed over 700 000 fresh and frozen ART cycles. Despite this, clinical embryology is only recognized as an official profession in 3 out of 27 national health systems. In most countries clinical embryologists need to be registered under another profession, and have limited possibilities for organized education in clinical embryology. Mostly they are trained for practical work by senior colleagues. ESHRE embryologist certification so far constitutes the only internationally recognized qualification; however this cannot be considered a subspecialization. LIMITATIONS, REASONS FOR CAUTION: Data were obtained through different methods, by involving national embryologist societies and cycle registers, collecting information from centre to centre, and in some cases by individual assessment of the situation. For these reasons, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: This paper presents the current status of clinical embryology and clinical embryologists in Europe and is an important step towards implementation of clinical embryology as an officially recognized profession. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: No.

An alternative real-time PCR method for the detection of thermotolerant Bacillus sensu lato contaminants in naturally-contaminated gelatine.

AIMS: Comparison of an internally-controlled real-time PCR assay with the current plate-based assay for the detection of Bacillus sensu lato contaminants in gelatine. METHODS AND RESULTS: A comprehensive TaqMan probe was designed allowing the real-time PCR assay to be fully inclusive for the gelatine-contaminating Bacillus s.l. species. An internal amplification control was implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (Kappa value = 0.94) was observed between the performance of the real-time PCR and the current plate-based method on naturally contaminated gelatines (n = 162). Relative accuracy, relative sensitivity and relative specificity were 97.5%. CONCLUSIONS: The real-time PCR assay is an adequate alternative of the current plate-based assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h. The gelatine-producing industry can ensure gelatine quality in a much faster way.

Biological Control of Gray Mold with Ulocladium atrum in Annual Strawberry Crops.

The efficacy of the fungal antagonist Ulocladium atrum to control gray mold in annual strawberry crops using waiting-bed transplants under field conditions was investigated. Seven field experiments were conducted with strawberry cv. Elsanta during the summer seasons of 1996-99 in the Netherlands. Treatments included untreated controls, fungicide programs, U. atrum spray programs, and crop sanitation. Under low disease pressure, U. atrum spray programs effectively reduced gray mold at harvest in four of seven experiments. Sprays of U. atrum starting at transplanting resulted in better control of gray mold than sprays starting at the beginning of flowering in only one of five experiments. Removal of necrotic leaves did not affect the level of gray mold, which demonstrated that strawberry leaves were not a significant inoculum source for Botrytis cinerea in this annual cropping system. These results suggest that U. atrum can be effective in reducing gray mold in strawberry crops, and further studies on the use of the antagonist in annual systems should consider flowering time as the best period to apply this antagonist.

The accuracy of physical diagnostic tests for assessing meniscal lesions of the knee: a meta-analysis.

OBJECTIVE: Our systematic review summarizes the evidence about the accuracy of those tests. SEARCH STRATEGY: We performed a literature search of MEDLINE (1966-1999) and EMBASE (1988-1999) with additional reference tracking. SELECTION CRITERIA: Articles written in English, French, German, or Dutch, that addressed the accuracy of at least one physical diagnostic test for meniscus injury with arthrotomy, arthroscopy, or magnetic resonance imaging as the gold standard were included. We excluded studies if no reference group or only test-positives had been included, if the study pertained to cadavers only, or if only physical examination under anesthesia was considered. DATA COLLECTION/ANALYSIS: Two reviewers independently selected studies, assessed the methodologic quality, and abstracted data using a standardized protocol. We calculated sensitivity, specificity, and likelihood ratios for each test, and summary estimates when appropriate and possible. MAIN RESULTS: Of 402 identified studies, 13 met the inclusion criteria. The results of the index and reference tests were assessed independently (blindly) of each other in only 2 studies, and in all studies verification bias seemed to be present. The study results were highly heterogeneous The summary receiver operating characteristic curves of the assessment of joint effusion, the McMurray test and joint line tenderness indicated little discriminative power for these tests. Only the predictive value of a positive McMurray test was favorable. CONCLUSIONS: The methodologic quality of studies addressing the diagnostic accuracy of meniscal tests was poor, and the results were highly heterogeneous. The poor characteristics indicate that these tests are of little value for clinical practice.

Insulin-like growth factor (IGF) binding proteins modulate the glucocorticoid-dependent biological effects of IGF-II in cultured fetal rat hepatocytes.

The role of insulin-like growth factor binding proteins (IGFBPs) in regulation by IGF-II of glycogenesis and DNA synthesis was investigated in hepatocytes isolated from fetal rat livers at days 15 and 18 of gestation and grown in the presence or absence of cortisol. IGFBP-1 was clearly revealed by Western ligand blot and immunoblot analysis of IGFBPs secreted into conditioned media. Its production and cellular messenger RNA (mRNA) were positively regulated by cortisol and increased in older cells. In the absence of IGFBP (fresh medium), glycogenesis, and DNA synthesis were stimulated by IGF-II and insulin. In each case, cortisol enhanced this stimulation. In the presence of IGFBPs (cell-conditioned media), IGF-II stimulation of DNA synthesis and to a lesser extent glycogenesis was inhibited. The degree of inhibition was directly related to IGFBP-1 production. IGFBPs had no effect on stimulation of glycogenesis and DNA synthesis by des(1-6)IGF-II, a structural analog of IGF-II that does not bind to IGFBPs. Insulin, whose biological effects were not modified by conditioned media, inhibited IGFBP-1 production. Comparison of the dose dependence of the two bioactivities showed that DNA synthesis was more sensitive to IGF-II than glycogenesis. Our results suggest that in the case of DNA synthesis the effects of IGF-II are mediated via the IGF-I receptor and those of insulin via the insulin receptor, whereas in the case of glycogenesis both are mediated via the insulin receptor. In conclusion, IGF-II and insulin stimulation of glycogenesis and DNA synthesis in cultured fetal hepatocytes depends on the presence of glucocorticoid and the stage of development. IGF-II action is negatively regulated by IGFBP-1 whose synthesis increases in the presence of glucocorticoids.

Gap junctional communication and regulation of the glycogenic response to insulin by cell density and glucocorticoids in cultured fetal rat hepatocytes.

Cell culture studies have revealed that metabolic functions of the adult hepatocyte are related to cell density. Development of the glycogenic response to insulin under glucocorticoid control was investigated in 15- and 18-day-old fetal rat hepatocytes plated at different cell densities. After culturing for 48 hours with glucocorticoids, the stimulatory effect of insulin on [14C]glucose incorporation into glycogen after 3 hours progressed from weak response (less than 1.4-fold) in sparse cultures to a maximal response in dense ones (3.0- to 4.5-fold), depending on the fetal stage. The response was always no more than 2.0-fold in the absence of glucocorticoids, even with dense cultures. Such a dual regulation pattern was not found for the glycogenolytic effect of glucagon similarly expressed regardless of culture conditions. When cells were clustered in limited circular regions of the dish, the insulin response was higher than for sparse cultures for a similar number of cells per culture. Using the scrape-loading technique with Lucifer Yellow CH, a positive dye transfer was obtained in clustered cultures providing that they were grown in the presence of glucocorticoids; insulin as well as glucagon stimulated twofold intercellular communication. Connexin32 (Cx32) and connexin26 (Cx26) protein levels were assayed by Western immunoblotting and developed according to age and exposure to glucocorticoids. Thus, glucocorticoids through development of gap junctions enabled establishment of intercellular communication that could be stimulated by insulin and glucagon in cultured fetal hepatocytes. Gap junction functioning and the biologic effect of insulin correlated closely.

Quantification of Mycelium of Botrytis spp. and the Antagonist Ulocladium atrum in Necrotic Leaf Tissue of Cyclamen and Lily by Fluorescence Microscopy and Image Analysis.

ABSTRACT A technique was developed to localize and quantify the internal mycelial colonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. This technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a monoclonal antibody specific for Botrytis spp. And an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was used to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hyphae of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume of each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and allowed the design of efficient sampling strategies for future experiments.

Time-dependent effects of insulin on lipid synthesis in cultured fetal rat hepatocytes: a comparison between lipogenesis and glycogenesis.

The lipogenic effect of insulin was studied in 18-day-old fetal rat hepatocytes after 2 to 3 days of culture in the presence of glucocorticoids when an acute stimulatory effect of insulin on glycogenesis was present. The rate of [1-14C]-acetate incorporation into lipids measured for 4 hours was much higher than with [U-14C]-glucose (30 v 3.8 nmol/h/mg protein). The stimulatory effect of insulin on lipid labeling remained weak (1.2-fold) and contrasted with its striking stimulatory effect on [U-14C]-glucose incorporation into glycogen (fourfold). When lipid labeling was assessed in longer experiments, increasing acetate concentrations in the medium stimulated the incorporation rate of [1-14C]-acetate into lipids (3.5-fold from 1 to 5 mmol/L after 36 hours) and decreased that of [U-14C]-glucose (by twofold). The stimulatory effect of insulin on the rate of lipid labeling developed with both precursors from 12 to 36 hours after insulin exposure (by approximately twofold) independently of acetate concentration and was not glucocorticoid-dependent, contrary to the glycogenic response. Addition of a glucose, load simultaneously with insulin increased the stimulation of lipogenesis when measured with [U-14C]-glucose (twofold to 3.7-fold). Besides contributing to an accumulation of larger and numerous lipid droplets in the cells, insulin increased fatty acid synthase activity by 26%, whereas malic enzyme was not affected. Thus, insulin-dependent lipogenesis in cultured fetal hepatocytes appears to be mostly regulated by a long-term mechanism, contrary to the glycogenic effect of insulin.

Sequential insulin degradation in cultured fetal hepatocytes in relation to chloroquine-dependent events.

Insulin cellular degradation was studied in cultured 18-day-old fetal rat hepatocytes in the presence and absence of insulin degradation inhibitors that decrease the glycogenic response to insulin. After cell incubation with 3 nM [125I]A14 or -B26 insulin, hormone degradation products associated with cells or present in the medium were analyzed by high-performance liquid chromatography. Within cells, four components containing intact [125I]A14 insulin A-chain and part of the B-chain (A1-A4, according to increasing retention times) were found together with two [125I]B26 insulin B-chain COOH-terminal fragments (B1 and B2). Medium degradation intermediates comprised B1 and B2 but not A1-A4. Cellular insulin fragments A3 and B2 exhibited a maximal transient accumulation after 2 min, whereas the others increased progressively to plateau after 10 min. Chloroquine inhibited the formation of A1, A2, and B1 by 70-80%, whereas that of A3, A4, and B2 was not significantly affected. N-ethylmaleimide and bacitracin, two inhibitors of insulin-degrading enzyme (IDE), decreased the formation of chloroquine-dependent cellular peptides. Thus cell-associated insulin degradation implied primarily two cleavages in B-chain near the COOH-terminus, the one sensitive to chloroquine and IDE inhibitors occurring after endosomal segregation of insulin and its receptor.

Role of beta-GP-derived Pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells.

The permissive effect of beta-GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto-ALP at physiological pH range. Beta-GP present in the culture medium for 8 days exerted a stimulatory effect on 45Ca incorporation into matrix cell layers while the ecto-ALP activity level measured on intact cells with a saturating concentration of pNPP was similar for cells grown either in the presence or absence of beta-GP. In both types of cultures, beta-GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of beta-GP on 45Ca incorporation and generation of Pi was similar (k phi = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and beta-GP hydrolyses, which were likely catalyzed by the same ecto-enzyme. The rate of 45Ca incorporation into matrix cell layers, which was high (0.90 mumol/4h/mg cell protein) in cells grown in the absence of beta-GP, was inhibited by 50% by levamisole. In cells grown in the absence of beta-GP, the 45Ca incorporation rate increased progressively after beta-GP addition, reaching after 12 h the value of cultures grown in the presence of beta-GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of beta-GP-derived Pi rapidly led to high 45Ca incorporation rate which was unaffected by levamisole. beta-GP removal from cultures grown in its presence reduced by 50% the 45Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto-ALP to hydrolyze beta-GP in cultured fetal calvaria cells, yet it influenced the beta-GP-stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels.

Role of Hsp70 synthesis in the fate of the insulin-receptor complex after heat shock in cultured fetal hepatocytes.

The influence of a mild heat shock on the fate of the insulin-receptor complex was studied in cultured fetal rat hepatocytes whose insulin glycogenic response is sensitive to heat [Zachayus and Plas (1995): J Cell Physiol 162:330-340]. After exposure from 15 min to 2 hr at 42.5 degrees C, the amount of (125)1-insulin associated with cells at 37 degrees C was progressively decreased (by 35% after 1 hr), while the release of (125)1-insulin degradation products into the medium was also inhibited (by 75%), more than expected from the decrease in insulin binding. Heat shock did not affect the insulin-induced internalization of cell surface insulin receptors but progressively suppressed the recycling at 37 degrees C of receptors previously internalized at 42.5 degrees C in the presence of insulin. When compared to the inhibitory effects of chloroquine on insulin degradation and insulin receptor recycling, which were immediate (within 15 min), those of heat shock developed within 1 hr of heating. The protein level of insulin receptors was not modified after heat shock and during recovery at 37 degrees C, while that of Hsp72/73 exhibited a transitory accumulation inversely correlated with variations in insulin binding, as assayed by Western immunoblotting from whole cell extracts. Coimmunoprecipitation experiments revealed a heat shock-stimulated association of Hsp72/73 with the insulin receptor. Affinity labeling showed an interaction between (125)1-insulin and Hsp72/73 in control cells, which was inhibited by heat shock. These results suggest that increased Hsp72/73 synthesis interfered with insulin degradation and prevented the recycling of the insulin receptor and its further thermal damage via a possible chaperone-like action in fetal hepatocytes submitted to heat stress.

Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells.

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6-8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (Ki = 45 microM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of 45Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to 45Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells.

Delayed glucocorticoid-dependent development of the insulin response after heat shock in cultured fetal hepatocytes: correlation with a transient defect in glucocorticoid receptor binding property.

The effects of a mild heat shock were investigated on glucocorticoid-induced maturation of the insulin glycogenic response and on glucocorticoid receptor (GR) in cultured 15-day-old fetal rat hepatocytes. In all experiments, cell heating at 42.5 degrees C from 30 to 90 min was applied at Day 1 of culture before exposure to glucocorticoids. In never heated cells, the insulin stimulation of glycogenesis, measured bh [14C]glucose incorporation into glycogen for 3 h, developed to be maximal after 32 h in the presence of 100 nM dexamethasone (2.4-fold). In cells preheated at 42.5 degrees C for times equal to or greater than 60 min before being returned to 37 degrees C in dexamethasone-containing medium, the insulin response was not seen after 32 h (1.3-fold versus 2.4-fold) but was clearly expressed after 48 h. Heat treatment induced a progressive decrease in intact cell GR binding, reaching 40% after 1 h. When cells were returned to 37 degrees C, GR binding following a lag time of 8 h increased up to complete restoration after 24 h. Such heat shock-induced variations affected the number of GR binding sites with little change in GR binding affinity, while no modifications were seen in the 97-kDa GR level from whole cell extracts as revealed by Western immunoblotting using an anti-GR antibody (BuGR2). [35S]Methionine metabolic labeling showed a reversible heat-stimulated synthesis of 70- and 90-kDa heat shock proteins (Hsps). Variations in Hsp90 level revealed by Western immunoblotting using an anti-Hsp90 antibody (AC88) were inversely correlated with time with GR binding. Therefore, a mild heat shock applied to cultured fetal hepatocytes led to a delayed development of the glycogenic response to insulin linked to a defect in GR binding property with no alteration in the GR protein level.

Regulation by insulin-like growth factor (IGF) binding proteins of IGF-II-stimulated glycogenesis in cultured fetal rat hepatocytes.

We previously showed that when added to fresh medium, insulin-like growth factor (IGF)-II stimulates glycogen synthesis in cultured 18-day-old fetal rat hepatocytes. In the present study, we investigated the influence of 24-h culture-conditioned medium on IGF-II- and insulin-induced glycogenesis. The stimulatory effect of IGF-II (2.9-fold) on [14C]glucose incorporation into glycogen over 3 h was dose dependently inhibited by conditioned medium, whereas that of insulin (3.2-fold) was unaffected. Western ligand and immunoblot analysis of the conditioned media revealed IGF binding protein (IGFBP)-1, IGFBP-2, and IGFBP-4, with a predominance of IGFBP-1. IGFBP-3 was not detected. Preincubation of conditioned medium with IGF-II for 4 h at 4 C restored the glycogenic effect of newly added IGF-II. Preincubation of fresh medium with recombinant IGFBP-1 or IGFBP-3 in the presence of IGF-II suppressed IGF-II stimulation of glycogen synthesis. IGFBPs alone had no effect on glycogenesis. Des(1-6)IGF-II and R6IGF-II, structural analogs of IGF-II that have weak affinity for the IGFBPs, elicited maximal stimulation, near that of IGF-II (2.8-fold and 3.1-fold vs. 3.0-fold for IGF-II), whether tested in fresh or conditioned medium. The inhibitory effect of conditioned medium on IGF-II-induced glycogenesis is therefore mediated by the IGFBPs via sequestration of IGF-II. This suggests that the IGFBPs, particularly IGFBP-1, produced by fetal rat hepatocytes in culture may play a role in regulating glycogenesis.

Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes.

The effects of a mild heat shock were investigated using cultured 15-day-old fetal rat hepatocytes in which an acute glucocorticoid-dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5 degrees C, cell surface [125I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37 degrees C, due toa decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [35S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73-kDa Hsc (heat shock cognate) and 72-kDa Hsp (heat shock protein). When cells were returned to 37 degrees C after 2 h at 42.5 degrees C, cell surface insulin binding showed a two-third restoration within 3 h (insulin receptor half-life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [14C]glucose incorporation into glycogen measured at 37 degrees C after 1- to 2-h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5-fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4-fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock-dependent events except for the enhanced basal glycogenesis. In insulin-unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress.

Protein kinase C and insulin receptor beta-subunit serine phosphorylation in cultured foetal rat hepatocytes.

In digitonin-permeabilized cultured foetal hepatocytes, insulin receptor beta-subunit was highly phosphorylated on serine residues in the presence of [gamma-32P]ATP and Ca2+, a process enhanced after short exposure to insulin with no detectable insulin receptor autophosphorylation. By contrast with this situation, experiments performed with isolated foetal insulin receptors revealed an insulin stimulation of both serine phosphorylation and tyrosine autophosphorylation. In permeabilized cells, insulin receptor beta-subunit phosphorylation was increased after a 2-min exposure to phorbol 12-myristate 13-acetate (PMA) prior to applying the permeabilization/phosphorylation step, while it was inhibited by chronic treatment with PMA leading to protein kinase C (PKC) down modulation. The PKC specific inhibitor, GF109203X, strikingly reduced basal and insulin-enhanced phosphorylation of insulin receptor beta-subunit in permeabilized cells, but failed to exert any effect with isolated receptors. Labelling of glycogen from [U-14C]glucose determined 1 h after a 10-min transitory exposure to insulin and/or modulators of PKC activity showed that PMA prevented insulin glycogenic response, whereas GF109203X was ineffective. Thus, although not directly responsible for insulin receptor serine phosphorylation in cultured foetal hepatocytes, PKC physiologically regulates this process which may inhibit insulin receptor tyrosine kinase activity. This regulation is independent of the antagonistic effect of PMA-activated PKC on insulin glycogenic response.

Insulin receptor beta-subunit serine phosphorylation in permeabilized cultured fetal rat hepatocytes.

Regulation of cellular protein phosphorylation by insulin was investigated after short exposure at 37 degrees C prior to applying the permeabilization/phosphorylation step in the presence of digitonin and [gamma-32P]ATP for 30 min at 4 degrees C. The results revealed major 32P incorporation into a limited number of membrane polypeptides exhibiting a molecular mass of 95, 58 and 51 kDa. Phosphorylation of 95 kDa protein was selectively inhibited with Ca(2+)-free EGTA-containing permeabilization/phosphorylation buffer and became predominant in the presence of Ca2+. Considering in particular its immunoprecipitation by a monoclonal antibody directed against insulin receptor, the 32P-labeled 95 kDa protein represented the beta-subunit of the insulin receptor. Its phosphorylation was transiently stimulated after exposure to insulin (35% after 2 min), and concerned mostly serine residues under both basal and stimulated conditions. Vanadate had a similar effect and both agents favored glycogenesis, whereas heparin which inhibited 95 kDa protein phosphoseryl phosphorylation had an opposite effect on glycogenesis. These results suggest a biological role for the membrane-associated phosphoseryl-protein kinase(s) and phosphatase(s) acting on the insulin receptor beta-subunit in cultured fetal hepatocytes.

Consumption and production of amino acids by insulin-responsive cultured fetal rat hepatocytes: the particular case of serine.

The ability for 18-day fetal rat hepatocytes in primary culture to modify extracellular amino acid concentrations was studied between 24 and 48 h of culture. Most of the 19 amino acids tested were found to be taken up by the hepatocytes. However, serine and glutamate appeared in the 24-hour-conditioned medium to be twice as concentrated as in the fresh medium. The profile of net consumption or production of amino acids was unchanged when the medium was supplemented with essential amino acids. The use of [U-14C]glucose revealed that serine released in the medium was mainly formed from glucose. The presence of insulin (10 nM) did neither significantly modify the variations of amino acid concentrations in the medium nor 2-amino[1-14C]isobutyric acid uptake by the cells, while the hormone produced a 2-fold increase in glycogen labeling from [U-14C]glucose. This study revealed that whatever the regulatory culture conditions considered a net serine production out of the cells occurred, which appears to be specific to the fetal stage.

Variations in the antagonistic effects of insulin and glucagon on glycogen metabolism in cultured foetal hepatocytes.

The antagonistic effects of insulin and glucagon on glycogen formation and mobilization were studied in cultured 18-day foetal rat hepatocytes with regard to different modes of exposure. Hormone combinations were achieved with a constant dose of 10 nM-insulin (maximal for the glycogenic effect of this hormone) and increasing doses of glucagon [from 0.03 to 10 nM: concn. causing half-maximal response (ED50) = 0.3 nM)]. When insulin and glucagon were added simultaneously, increasing glucagon concentrations progressively depressed the glycogenic effect of insulin and 0.3 nM-glucagon antagonized the insulin effect completely. The maximal glycogenolytic effect of glucagon was observed at concentrations greater than 1 nM. When the two hormones were introduced successively, with an interval of 4 h between additions, the effect of the second hormone was always fully expressed between 4 and 8 h. at which time the effect of the first hormone had ceased; the dominance of glucagon over insulin was also lost, due to cell desensitization to glucagon. Both continuous or intermittent (10 min on/10 min off periods) exposure to insulin and/or glucagon gave similar antagonistic effects, while in cells exposed to insulin plus glucagon alternating with exposure to insulin or glucagon alone, the glycogenic effect of insulin was less or more antagonized respectively by glucagon. Whatever the situation, the results obtained could not be related to antagonism by a glucagon-induced rise in either cyclic AMP levels (ED50 = 3 nM) or cell-surface hormone binding. Thus, depending on the hormonal state and the mode of hormone administration, regulation of glycogenesis in cultured foetal hepatocytes appears to be different from that predicted by the insulin/glucagon molar ratio, which is strikingly altered in the perinatal period.

Role of serine biosynthesis and its utilization in the alternative pathway from glucose to glycogen during the response to insulin in cultured foetal-rat hepatocytes.

The role of serine as a possible intermediate of the alternative pathway from glucose to glycogen was investigated under basal and insulin-stimulated conditions in 18-day cultured foetal-rat hepatocytes because these cells cannot use pyruvate-derived metabolites [Bismut & Plas (1989) Biochem. J. 263, 889-895]. Incubation of cells with [U-14C]glucose for 24 h led to a release of labelled serine in the medium concomitantly with a net serine production (100 nmol/24 h per culture). The rate of [14C]serine formation (close to 3 nmol/h per culture) indicated that a large part of newly formed serine originated from glucose. When short-term experiments were performed at day 2, glycogen labelling from [U-14C]serine or [U-14C]glycine, which was increased 3-fold by insulin after 2 h, evidenced their participation as glycogenic precursors. When a double-isotope procedure with [U-14C,3-3H]glucose was used, the direct and the alternative pathways from glucose were found to contribute to glycogenesis by 75 and 25% respectively. Cycloserine (18 mM), a transaminase inhibitor, strongly inhibited glycogen labelling from [U-14C] serine while producing a 70% increase in glucose incorporation by the alternative pathway, in both the presence and the absence of insulin. The inhibitor had no effect on the direct pathway from glucose to glycogen. Supplementation with 1 mM-hydroxypyruvate, a serine-derived metabolite, did not affect direct glucose incorporation, whereas the alternative pathway was stimulated whether insulin was present or not. These results indicate that the sequence glucose----serine----glycogen is operative in cultured foetal hepatocytes. The alternative pathway interferes with hydroxypyruvate utilization, and is likely mediated by the serine aminotransferase pathway, independently of the acute glycogenic action of insulin.