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.

Differences between glucose and insulin stimulation of glycogenesis in cultured fetal hepatocytes.

The glycogenic effects of a glucose load (15 mM) and/or insulin (10 nM) were studied in 18-day-old fetal rat hepatocytes after 2 days of culture when medium contained 4 mM glucose. A glucose load led to a stimulation of [14C]glucose glycogen labelling (20 min) earlier than with insulin (30-40 min); maximal stimulations were 3-fold after 1 h for the glucose load and 5-fold after 2-3 h for insulin. Simultaneous addition of the two agents produced synergic effects. When insulin was added 4 h after a glucose load (or vice versa), a second glycogenic response was elicited: a further addition of the same glycogenic agent was ineffective. The early glycogenic effects (up to 2 h) also occurred in the presence of 10 microM cycloheximide, with, however, some decrease of insulin stimulation. The contribution of medium glucose to the glycogen formed for 2 days (67% in the absence of glycogenic agent) was clearly enhanced by a glucose load and to a lesser degree by insulin after a 4-h exposure (83 and 71%, respectively). This was accompanied by a related modification of the participation of glucogenic precursors such as fructose and galactose. Thus, acute glycogenic response to glucose and insulin appeared both synergic and independent, and quite different in several aspects in cultured fetal hepatocytes.

Dexamethasone binding sites and steroid-dependent stimulation of glycogenesis by insulin in cultured fetal hepatocytes.

The binding of [3H]dexamethasone and the effect of insulin on [14C]glucose incorporation into glycogen were studied in cultures of fetal rat hepatocytes transplanted from 15 and 18 days of gestation, i.e. before and just at the critical stage of glucocorticoid-dependent maturation of the rat fetus. Both types of cell cultures contained approximately 50,000 specific glucocorticoid receptors per cell, with an affinity of 6 nM. Glycogenesis was hardly stimulated by insulin at the time of transplantation, especially in 15-day-old fetal hepatocytes. The stimulatory effect of insulin increased in the presence of dexamethasone (100 nM) to reach, after 40 h of treatment, 270% and 440% of the control values in 15- and 18-day-old fetal hepatocytes, respectively. A shortening of the exposure time to steroid necessary to trigger the insulin response was observed with 18-day-old cells (20 h). The half-maximal insulin-induced stimulation of glycogenesis was obtained with a lower concentration of dexamethasone in 18-day-old than in 15-day-old hepatocytes (3.2 +/- 0.32 vs. 7.9 +/- 0.29 nM, n = 5; P less than 0.001). Although high affinity dexamethasone binding sites exist in fetal rat hepatocytes before the critical stage of glucocorticoid influences, some maturation occurs between the 15th and the 18th day of gestation, which is associated with an increase in cell sensitivity to dexamethasone and in the amplitude of the steroid-induced glycogenic response to insulin.

Interaction of glucagon and epinephrine in the regulation of adenosine 3',5'-monophosphate-dependent glycogenolysis in the cultured fetal hepatocyte.

The hormonal control of glycogenolysis has been studied in 3-day-cultured fetal rat hepatocytes which contained stored glycogen. A single addition of 10 nM glucagon or 10 nM epinephrine produced an identical maximal glycogenolytic response, which developed within 4 h and ceased thereafter. The amount of glycogen degraded represented 60% of the stored glycogen or 95% of the newly synthesized glycogen after a 4-h preincubation period in the presence of [14C]glucose. The latter result demonstrates that both hormones interact on the same hepatocytes. Stimulation of glycogenolysis by glucagon or or epinephrine was preceded by an accumulation of intracellular cAMP. From the decreasing order of potency of isoproterenol, epinephrine, norepinephrine, and phenylephrine to activate glycogenolysis, it can be concluded that the epinephrine effect is mainly mediated by beta-adrenergic receptors. When glucagon and epinephrine were added simultaneously at maximal concentrations, the glycogenolytic effects were not additive. Moreover, when epinephrine was added 4 h after glucagon, it elicited a second glycogenolytic response, so that the amount of glycogen degraded represented 80% of the stored glycogen. At this stage, a second addition of glucagon was ineffective, and the extent of the glucagon-induced loss of response depended on the size of the first dose of hormone. Cell densensitizatin to glucagon for glycogenolysis was closely related to the associated response in cAMP production. This desensitization was found to be highly specific for glucagon and was accompanied by a defect in specific glucagon binding. The occurrence of a specific negative regulation of the response to glucagon explained how epinephrine was able to mobilize glycogen accumulated in the continued presence of glucagon during hepatocyte development in culture.

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.

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.

Effects of modified insulin B21-B26 fragments on glycogenesis and on insulin-receptor complex fate in cultured fetal hepatocytes.

Biological activity and interference with insulin receptor complex fate of two modified sequences of insulin B21-B26, beta-Ala-Arg-Gly-Phe-Phe-Tyr-NH2 (DP-432) and beta-Ala-Arg-Pro-Phe-Phe-Tyr-NH2 (DP-640), were studied in cultured 18-day-old fetal rat hepatocytes known to respond to insulin by an acute stimulation of glycogenesis. The two derivatives stimulated [14C]glucose incorporation into glycogen in the absence of insulin independently of the deprivation of serum in the medium. The maximal effect of 3 mM DP-640 after 2 h, more pronounced than with 3 mM DP-432, was of the same order as that obtained with 10 nM insulin alone (stimulation index: 4.7 +/- 0.7, 2.5 +/- 0.2 and 3.6 +/- 0.9, n = 4, with DP-640, DP-432 and insulin, respectively) whereas insulin B-chain decreased glycogen labeling. Simultaneous addition of derivatives and insulin at maximal concentrations produced nearly additive effects. DP-640, as well as DP-432, increased the amount of [125I](A14) or (B26) human insulin associated with cells at 37 degrees C and inhibited intracellular insulin degradation with differences depending on the kind of insulin isomer and derivative, while the rapid insulin receptor cycle was not affected. Thus, the two derivatives specifically modified the cellular processing of insulin in cultured fetal hepatocytes, and exerted an insulin-like effect on glycogenesis clearly enhanced through modification of DP-432 by substitution of glycine for proline (DP-640).

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.

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.

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.

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.

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.

[Proceedings: Cortisol-dependent glycogen storage and responses to insulin and glucagon in rat fetal cultured hepatocytes (author's transl)]. / Rôle du cortisol sur le développement de la fonction glycogénique du rat et sa régulation dans l'hépatocyte foetal en culture

It has been shown that in primary cultures of rat fetal hepatocytes cortisol induces the development of glycogen storage ability. Cortisol has also a permissive effect in insulin action on the development of the glycogen synthetic pathway. However the regulation of glycogenolytic pathway by glucagon, which is present before any significant amount of glycogen storage, is not cortisol dependent.

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.

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.

Glucocorticoid regulation of chloroquine nonsensitive insulin degradation in cultured fetal rat hepatocytes.

The influence of cortisol and other culture conditions on insulin degradation by the chloroquine-sensitive pathway and the chloroquine-nonsensitive pathway (CNP) was investigated in fetal rat hepatocytes during 3 days of culture. The proportions of the chloroquine nonsensitive release of 125I-insulin degradation products into the conditioned medium/h increased from the 1st to the 3rd day of culture, i.e. from 19 to 50% by cells grown in the presence of cortisol and from 17 to 82% by those grown in the absence of cortisol. Replacement of the conditioned medium with the respective fresh medium dramatically enhanced cellular insulin degradation by CNP, i.e. from 22 to 58%, and 19 to 85% in cells grown for 2 days in the presence and absence of cortisol, respectively. Thus, the conditioned medium contained some factor(s) that inhibited CNP. Therefore, we used the inhibited insulin and alpha-casein degradation by papain in vitro as an assay to investigate the nature of the putative anti-(insulin) protease. Cycloheximide completely prevented the appearance of anti-papain activity in the medium. Conditioned medium obtained from cells grown in the presence of cortisol contained about 2-fold more anti-papain activity than the medium that was obtained in the absence of the steroid. The release of anti-papain activity also declined with time from 1 to 3 days of culture and showed an inverse relationship with the magnitude of cellular insulin degradation by CNP. The inhibition of papain-mediated insulin degradation by the anti-(insulin) protease was noncompetitive. The anti-(insulin) protease was nondialyzable (up to the 10-kDa exclusion limit) and inactivated by heat treatment at 50 degrees C for 30 min. These results suggest that fetal hepatocytes synthesize and secrete a glucocorticoid-regulated heat-labile low molecular mass (less than 25 kDa) anti-(insulin) protease, which may contribute to the suppression of insulin degradation caused by the enzymes involved in CNP.

Pathways of glycogen synthesis from glucose during the glycogenic response to insulin in cultured foetal hepatocytes.

The pathways of glycogen synthesis from glucose were studied using double-isotope procedures in 18-day cultured foetal-rat hepatocytes in which glycogenesis is strongly stimulated by insulin. When the medium containing 4 mM-glucose was supplemented with [2-3H,U-14C]glucose or [3-3H,U-14C]glucose, the ratios of 3H/14C in glycogen relative to that in glucose were 0.23 +/- 0.04 (n = 6) and 0.63 +/- 0.09 (n = 8) respectively after 2 h. This indicates that more than 75% of glucose was first metabolized to fructose 6-phosphate, whereas 40% reached the step of the triose phosphates prior to incorporation into glycogen. The stimulatory effect of 10 nM-insulin on glycogenesis (4-fold) was accompanied by a significant increase in the (3H/14C in glycogen)/(3H/14C in glucose) ratio with 3H in the C-2 position (0.29 +/- 0.05, n = 6, P less than 0.001) or in the C-3 position (0.68 +/- 0.09, n = 8, P less than 0.01) of glucose, whereas the effect of a 12 mM-glucose load (3.5-fold) did not alter these ratios. Fructose (4 mM) displaced [U-14C]glucose during labelling of glycogen in the presence and absence of insulin by 50 and 20% respectively, and produced under both conditions a similar increase (45%) in the (3H/14C in glycogen)/(3H/14C in glucose) ratio when 3H was in the C-2 position. 3-Mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis from lactate/pyruvate, further decreased the already poor labelling of glycogen from [U-14C]alanine, whereas it increased both glycogen content and incorporation of label from [U-14C]serine and [U-14C]glucose with no effect on the relative 3H/14C ratios in glycogen and glucose with 3H in the C-3 position of glucose. These results indicate that an alternative pathway in addition to direct glucose incorporation is involved in glycogen synthesis in cultured foetal hepatocytes, but that insulin preferentially favours the classical direct route. The alternative foetal pathway does not require gluconeogenesis from pyruvate-derived metabolites, contrary to the situation in the adult liver.

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.