Influence of environmental factors on the metabolism of glucose by preimplantation mouse embryos in vitro

The metabolism of glucose by late preimplantation mouse embryos was studied in a variety of media whose composition had been changed to reflect the environmental conditions in the uterus more closely than do standard culture media. The effects of combinations of energy substrates, the presence or absence of amino acids and the level of potassium in the medium were investigated. The use of energy substrates for in vitro culture at levels present in the uterine environment resulted in rates of synthesis and degradation of glycogen pools similar to those obtained using standard in vitro culture conditions but elevated incorporation into non-glycogen macromolecules. Amino acids influenced the metabolism of glucose by limiting the entry of glucose carbon into the non-glycogen macromolecular pool and directing more glucose into the synthesis of acid-soluble glycogen. Increasing the K+ concentration to 60 mM in the culture medium caused a small but significant increase in the number of eight-cell embryos degenerating during culture for 24 h but the metabolism of glucose was unaffected over this time. At the time of morula transformation to the blastocyst this level of potassium ions suppressed glycogen synthesis by 50% over 5 h but did not affect its turnover during chase culture. It is concluded that factors other than those studied here contribute to the maintenance of the low glycogen levels found in uterine embryos.     

Effects of prostaglandins E-2 and F-2 alpha on the metabolism of [U-14C]glucose by mouse morulae-early blastocysts in vitro

During 5-h culture in the presence of radioactive glucose, PGE-2 (10 micrograms/ml) significantly inhibited incorporation of glucose into the acid-soluble glycogen pool. PGE-2 at 1 and 10 micrograms/ml and PGF-2 alpha at 1 microgram but not 10 micrograms/ml stimulated incorporation of glucose into non-glycogen macromolecules during culture. However, the utilization of acid-soluble glycogen and other biochemical pools was not affected by the presence of PGs in the medium during 24-h chase culture of pulse-labelled embryos. Carbon dioxide production was significantly suppressed in the presence of PGs but accumulation of lactate was not affected. The results indicate that PGE-2 and PGF-2 alpha, in physiological concentrations, directly influence the metabolism of glucose by preimplantation embryos.     

Effects of oestradiol and progesterone on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro

The addition of progesterone (10(-7) to 10(-5) M) and/or oestradiol (10(-10) M) during 24-h chase culture of pulse-labelled morulae-early blastocysts did not affect the degradation of radiolabelled glycogen or other biochemical fractions. The presence of a high concentration of progesterone (10(-5) M) during 5-h pulse culture significantly inhibited incorporation of substrate carbon from [U-14C]glucose into both the acid-soluble and acid-insoluble glycogen fractions, but had no effect on non-glycogen fractions. Catabolic utilization of glucose as estimated by the rate of carbon dioxide and lactate production was not affected by the presence of progesterone (10(-7) to 10(-5) M), oestradiol (10(-10) to 10(-8) M) or a combination of both. The results indicate that ovarian steroids at expected physiological concentrations do not directly influence embryonic energy metabolism.     

Effects of coculture with uterine epithelial cells on the metabolism of glucose by mouse morulae and early blastocysts

Coculture of mouse morulae/early blastocysts with isolated endometrial epithelial cells reduced incorporation of glucose carbon into embryonic glycogen but had no significant effect on incorporation into other internal carbon pools during a 5-h culture in serum-supplemented Dulbecco's modification of Eagle's minimum essential medium. Turnover of glycogen pools during 24-h chase culture of pulse-labelled embryos was unaffected by the presence of uterine epithelial cells recovered from day-4 pregnant or non-pregnant mice. However, significantly more label was retained in non-glycogen macromolecules during chase in the presence of endometrium recovered from non-pregnant than from pregnant uteri.     

Peptidoglycan turnover during growth of a Bacillus megaterium Dap- Lys- mutant.

The aim of this study was to ascertain whether or not the absence of cell wall growth zones, deduced from the analysis of autoradiographs of DL-[3H]mesodiaminopimelic acid pulse-labeled cells of a Dap- Lys- mutant of Bacillus megaterium, was due to a high peptidoglycan turnover. Turnover was determined in very precise experimental conditions because two kinds of turnover occurred: a low, acid-soluble turnover and a high, acid-insoluble one. The latter was detected during a chase in the culture medium when bacteria were centrifuged before treatment with trichloroacetic acid. Otherwise the acid-insoluble released material precipitated with the bacteria. In the electron microscope this material presented a globular structure and contained both peptidoglycan and teichoic acid. The acid-insoluble turnover was mainly produced by a lytic acitivity that was released into the culture medium. This thermolabile activity was not due to cell lysis. It was implicated in septum cleavage and in the detachment of wall fragments from the cell surface, but did not seem indispensable for cell elongation. The acid-soluble turnover was much weaker and seemed to be indispensable for cell elongation.     

Metabolism of glucose by human embryos

Glucose turnover, as measured by CO2 production, lactate accumulation and carbon incorporation from [U-14C]glucose as sole energy substrate, was low on the 2nd day of culture of human embryos resulting from in-vitro fertilization but above that of unfertilized oocytes. In general, all parameters of metabolism increased substantially during the following 2 days of development but the rate of increase in lactate production was greater than that of CO2, especially between Days 3 and 4. Within developing embryos, no correlation was evident between the metabolic turnover of glucose and the method of patient stimulation, the morphological quality of embryos or the apparent rate of cleavage in culture. The results indicate that, before Day 3 of development, glucose is not effective as an energy source for the human embryo because of a blockade to glycolysis similar to that in mouse embryos.     

In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos

The glucose metabolism and embryonic development of rat embryos during organogenesis was studied using embryo culture. Glucose uptake and embryonic growth and differentiation of 10.5-day explants (embryos + membranes) were limited by the decreasing glucose concentration, but not the increasing concentration of metabolites, in the culture media during the second 24 h of a 48 h culture. No such limitations were found on the embryonic development of 9.5-day explants during a 48 h culture although glucose uptake was slightly reduced at very low concentrations of glucose. From the head-fold stage to the 25-somite stage of development, glucose uptake was characteristic of the stage of development of the embryo and not the time it had been in culture. Embryonic growth of 9.5-day explants was similar to that previously observed in vivo. Glucose uptake by 9.5-day explants was dependent on the surface area of the yolk sac and was independent of the glucose concentration in the culture media (within the range of 9.4 to 2.5 mM). The proportion of glucose converted to lactate was 100% during the first 42h of culture then fell to about 50% during the final 6h. The protein contents of both the extraembryonic membranes and the embryo were dependent on the glucose uptake.     

Incorporation of (U-14C)-glucose into glycogen in normal rat pancreatic islets

The incorporation of glucose into glycogen was determined in pancreatic islets isolated from normal rats and incubated with glucose (5 or 20 mM) and compounds known to affect glycogen metabolism in other tissues. Incubation of pancreatic islets with glucose (20 mM) induced a marked increase in radioactive glycogen. Exposure to epinephrine in the presence of glucose (20 mM) slightly increased incorporation of glucose into glycogen. In contrast the incorporation of glucose into glycogen was not affected when isolated islets were exposed to glucagon or insulin, whereas anti-insulin serum in the incubation medium decreased radioactive glycogen formation.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle effect of insulin,epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigared in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 nM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5–10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures.

Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle. Effect of insulin, epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigated in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 mM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5-10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Role of glucose in cloned mouse embryo development

Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early beneficial effect during the first cell cycle. This early beneficial effect of glucose is not displayed by parthenogenetic, fertilized, or tetraploid nuclear transfer control embryos, indicating that it is specific to diploid clones. Precocious localization of the glucose transporter SLC2A1 to the cell surface, as well as increased expression of glucose transporters and increased uptake of glucose at the one- and two-cell stages, is also seen in cloned embryos. To examine the role of glucose in early cloned embryo development, we examined glucose metabolism and associated metabolites, as well as mitochondrial ultrastructure, distribution, and number. Clones prepared with cumulus cell nuclei displayed significantly enhanced glucose metabolism at the two-cell stage relative to parthenogenetic controls. Despite the increase in metabolism, ATP content was reduced in clones relative to parthenotes and fertilized controls. Clones at both stages displayed elevated concentrations of glycogen compared with parthenogenetic controls. There was no difference in the number of mitochondria, but clone mitochondria displayed ultrastructural alterations. Interestingly, glucose availability positively affected mitochondrial structure and localization. We conclude that cloned embryos may be severely compromised in terms of ATP-dependent processes during the first two cell cycles and that glucose may exert its early beneficial effects via positive effects on the mitochondria.     

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: Influence of the maternally inherited components

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose. © 1996 Wiley-Liss, Inc.     

Peptidoglycan turnover during growth recovery after chloramphenicol treatment in a Dap-Lys-mutant of Bacillus megaterium

The study of diaminopimelic acid (DAP) incorporation and turnover during growth recovery in chloramphenicol-treated (CMP-treated) Bacillus megaterium cells showed that two kinds of turnover occurred. A low acid-soluble turnover appeared as soon as growth resumed in bacteria labeled before the CMP treatment and at the middle of the first generation in those labeled during the treatment. The acid-insoluble turnover appeared only at the beginning of the second generation of growth resumption in bacteria labeled before CMP addition and at the beginning of the third generation in those labeled during the CMP treatment. The acid-soluble release observed during the period of cell wall thinning is too low to account for the decrease of the wall thickness and the acid-insoluble loss appears after this period. When bacteria were transferred into partially spent medium instead of fresh culture medium the acid-insoluble release started to appear half a generation sooner. Electron microscopic observations showed that in this case, large scales detached from the cell wall. This activity of wall degradation was not observed when the partially spent medium was previously heated for 10 min at 100 degree C. The persistence of a thick wall on cell ends during the first generation does not reflect an absence of growth sites because their labeling on autoradiographs is high. Rather, it seems to be due to a low lytic activity at the poles.     

Transient stimulation of glucose metabolism by insulin in the 1-day chick embryo

Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1-1.0 ng/ml) than AO (30%; 1-100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate). The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.     

Effects of insulin on glucose metabolism in isolated heart myocytes from adult rats

The study examined the effect of insulin on glucose metabolism in freshly isolated calcium-tolerant heart myocytes from adult rats. The uptake of 2-deoxyglucose demonstrated an initial lag in response to insulin and the maximal insulin effect was not attained until after 3 min preincubation with the hormone. A dose-response study of 14CO2 production from [14C]glucose revealed that the maximum insulin stimulation of glucose utilization occurred with 5 mU/ml. Both the uptake and the oxidation of glucose proceeded at a linear rate in the absence and presence of insulin. However, insulin exerted a greater effect on the uptake (42-54%) than on the oxidation (17-22%) of exogenous glucose. Incorporation of glucose into glycogen was markedly increased by insulin and resulted in the myocyte glycogen concentration returning to in vivo levels. In the absence of insulin, glucose incorporation plateaued within 10 min of incubation and the glycogen concentration was not altered. Our findings also indicate that at equilibrium, insulin-treated cells exhibited a higher glycogen turnover rate. It thus appears that insulin exerts a differential effect on the different pathways in glucose metabolism in the isolated cardiac cells. This may be related in part to their quiescent state and lower energy demand.     

Identification of Insulin in Chick Embryo Retina During Development and Its Inhibitory Effect on DNA Synthesis

Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid-insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time-course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2-6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was found with 10(-8) M insulin. Insulin caused similar effects also on thymidine kinase activity. All these effects were obtained by using minimal essential medium without glutamine. The addition of glutamine to the medium reduced the inhibitory effect of insulin. Retinas of chick embryos contain immunoreactive insulin. Retinal immunoreactive insulin was at the highest level (1.12 ng/mg of protein) in the youngest retinas studied (day 6), then it declined with age, reaching the lowest value (0.58 ng/mg of protein) at day 14. This value did not vary significantly during the third week of development. A potential biological role of insulin in retinal development is discussed.     

EFFECT OF INSULIN ON LEVELS AND TURNOVER OF INTERMEDIATES OF BRAIN CARBOHYDRATE METABOLISM IN VIVO

Abstract— –The rates of incorporation of ¹⁴C from [U-^l4C]glucose into intermediary metabolites have been measured in rat brain in vivo. The time course of labelling of glycogen was similar to that of glutamate and of glucose, which were all maximally labelled between 20 and 40min, but different from lactate, which lost radioactivity rapidly after 20min. The extent of labelling of glycogen (d.p.m./ μ mol of glucose) was of the same order as that of glutamate at 20 and 40 min after injection of [¹⁴C]glucose. However, calculations of turnover rates showed that glutamate turns over some 8-10 times faster than glycogen. Insulin, intracisternally applied, produced after 4-5 h a 60 per cent increase in glucose-6-P and a 50 per cent increase in glycogen. There was no change in the levels of glucose, glutamate or lactate, nor in the activity or properties of the particulate and soluble hexokinase of the brain. The injection of insulin affected neither the glycogen nor glucose contents of skeletal muscle from the same animals. The effects of insulin on the incorporation of ^l4C into the metabolites contrasted with its effects on their levels. The specific activities of glycogen and glucose were unchanged and there was a slight but non-significant increase in the specific activity of glutamate. The time course of incorporation into lactate was unaffected up to 20 min, but a significant delay in the loss of ¹⁴C after 20 min occurred as a result of the insulin injection. At 40 min, the specific activity of cerebral lactate was 60 per cent higher in insulin-treated animals than in control animals. The results are interpreted in terms of an effect of insulin on glucose uptake to the brain, with possibly an additional effect on a subsequent stage in metabolism, which involves lactate.     

Metabolic shift from lipogenesis to glycogenesis in the last instar larval fat body of the silkworm,Bombyx mori

When [¹⁴C]glucose was injected into the last instar larvae of the silkworm, Bombyx mori, the label was incorporated into various tissues at varying degrees depending on the developmental stages. Fat body exhibited high incorporation rates throughout the feeding periods. Silk glands became active in incorporation but midgut decreased toward larval maturation. The pulse labeling experiment clearly demonstrated that the metabolic shift from lipogenesis to glycogenesis occurred in fat body at the middle of the last instar; a predominant incorporation was found in lipids when [¹⁴C]glucose was injected at the early stage, while at the late stage glycogen synthesis became most active. Incorporation into fat body proteins was not a major factor throughout the instar. Extirpation of silk glands enhanced incorporation into glycogen and proteins at the late stage but did not affect lipid synthesis. Long-term chase showed that fat body lipids and proteins synthesized at the early stage were totally carried over into the pupal fat body, while much glycogen produced at the late stage was used during the larval-pupal transformation with the remainder carried over into the pupa.From these results the metabolic shift from lipogenesis to glycogenesis in fat body is discussed in relation to the storage function of the fat body for pupal metamorphosis.     

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.