Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos.     

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.     

M16添加牛磺酸和EDTA支持昆明白小鼠体外受精并发育至囊胚

在以往研究工作的基础上证明了通过添加2.5mmol/L的牛磺酸和0.1mmol/L的EDTA至M16培养液中,可支持昆明白小鼠的体外受精（IVF）,并支持受精卵突破2－细胞阻滞发育至囊胚期。本研究进一步证明了牛磺酸和EDTA在昆明小鼠早期胚胎发育和克服2－细胞阻滞中起关键作用。 Abstract:The results of our early experiments show that the 2-cell block can be overcome by culturing zygote in modified M16 ,modified CZB and TE medium.Our research shows that the taurine and EDTA play key role in overcoming 2-cell block in Kunming mouse.The results show that the addition of taurine and EDTA to M16 medium can support the IVF and develoment to blastocyst in vitro in Kunming strain mice.This is the first report that M16 medium added with taurine and EDTA can be used in both IVF and culture medium to overcome the 2-cell block of embryo development in Kunming strain mouse.     

Development of 1-cell embryos from different strains of mice in CZB medium

One-cell embryos from several different strains of mice have been cultured to the blastocyst stage in CZB medium. CZB medium can be used to culture CF1 x B6SJLF1/J 1-cell embryos to the blastocyst stage provided glucose is introduced into the medium on Day 3 of culture. The amount of glucose required for embryo development was titrated using a concentration range of 5.5 to 49.5 mM. With the exception of the highest concentration, all glucose levels tested supported 65-85% development to the morula and blastocyst stages. Variations of CZB medium were tested for their ability to support the development of 1-cell embryos from 4 strains of mice. For embryos from CF1 and DBA/2J (both x B6SJLF1/J) mice, which exhibit a "2-cell block" to development in vitro, CZB medium containing glutamine with the addition of glucose on Day 3 supported optimum development from the 1-cell stage to morula and blastocysts (79% and 87%). For embryos from B6D2F1/J and CD1 female mice (both x B6SJLF1/J males), which do not exhibit a "2-cell block" to in vitro development, optimum development to morula and blastocyst stages (95% and 50%) was in CZB medium containing both glutamine and glucose from the start of culture.     

Effects of the culture density of mouse zygotes on the development in vitro and in vivo

Different numbers of CD-1 mouse zygotes(1, 5, 10, 20, 40 and 60) were cultured in 10 mul M16 medium, in M16 medium+EDTA, in M16 dedium+SOD+thioredoxin, and in CZB medium, respectively. When the zygotes, regardless of the number, were cultured with M16, no blastocysts could be obtained. The suitable ratio of embryos to 1 mul of M16 medium+EDTA or M16 medium+SOD+thioredoxin was 1:1 or 2:1. Medium volume from 1 to 10 mul did not affect blastocyst development when the embryo density was 1:1. However, blastocysts obtained from zygotes cultured singly had fewer cell numbers and showed inferior development to live fetuses after transfer to recipients. When CZB medium was used, suitable embryo density was not clear. The ratio of embryos to volume of culture medium was shown to be an important factor for in vitro culture of mouse zygotes.     

Differential effect of hexoses on hamster embryo development in culture

The effects of glucose, fructose, and galactose on hamster embryo development in the absence of phosphate were studied in culture. One- and two-cell embryos were cultured to the blastocyst stage in HECM-9 medium without hexose or in medium with increasing concentrations of hexoses. Embryo development, cell number, and cell allocation were assessed in blastocysts. Blastocyst viability was determined by transfer to pseudopregnant recipients. Although 0.25 mM fructose increased mean cell number, low glucose concentrations had no stimulatory effect on development to blastocyst. Both galactose and 5.0 mM glucose were detrimental to embryos. Addition of 0.5 mM glucose increased implantation and fetal viability as compared with controls. Compared with 0.5 mM glucose, treatment with 0.25 mM fructose gave similar implantation and fetal viability, whereas 5.0 mM glucose tended to decrease implantation and significantly decreased fetal development. These data demonstrate that morphology is a poor indicator of embryo viability and that exposure of preimplantation embryos to glucose or fructose is important for embryo viability post-transfer. Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with fructose suggest that fructose may be more appropriate than glucose for inclusion in culture medium.     

Evidence for extracellular enzymic activity of the isolated perfused rat heart

1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of ¹⁴CO₂ from [U-¹⁴C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-¹⁴C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-¹⁴C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.     

Effect of a high fructose diet on the activities of enzymes implicated in the conversion of fructose to glucose by the liver and the intestinal mucosa of the rat

The activity of enzymes implicated in the metabolic pathway of fructose to glucose conversion was shown in rat liver and intestine. In rats on normal diet, the specific activity of glucose-6-phosphatase, fructokinase, fructose-1,6-diphosphatase and triokinase was low in the intestine confirming that sugar conversion is not operative in this organ. In rats on a fructose diet, all the specific enzymatic activities tested were increased except for the hepatic triokinase and triose phosphate isomerase and for the intestinal triose phosphate isomerase. The intestine acquires the possibility to transform fructose to glucose by modifying the activities of enzymes implicated in the same metabolic pathway as that intervening in the liver.     

One-minute exposure of 4-cell mouse embryos to glucose overcomes morula block in CZB medium

One-cell mouse embryos that block at the 2-cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1-min exposure to 27 mM glucose at the 4-cell stage (～42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67–73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts). © 1994 Wiley-Liss, Inc.     

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94-99%) and blastocyst (90-96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94-98%) and blastocysts (91-93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.     

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94–99%) and blastocyst (90–96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94–98%) and blastocysts (91–93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.     

克服昆明小鼠体外受精卵发育阻滞方法的研究

本研究应用CZB和WM培养液进行昆明小鼠体外受精胚胎的发育培养,建立了一个可行的胚胎体外培养的新方法,并通过改变培养液的成分及其含量,对胚胎发育的阻滞机理和突破方法进行了初步的探索。培养于WM中的受精卵发生阻滞,有48％停留于2细胞阶段;而CZB中的胚胎有81％发育为桑椹胚和囊胚。在WM中添加EDTA和谷氨酰胺得到了66％的囊胚;加大WM中乳酸钠和丙酮酸钠的比值未能克服发育的阻滞现象。实验结果表明,EDTA和谷氨酰胺在克服阻滞时具有协同作用。     

Use of a protein-free medium for the in vitro development of sheep embryos]

The employment of protein-free medium for the culture of ovine embryos collected at the 1-2 cell stage from superovulated ewes was investigated. For this purpose sheep zygotes were randomly allocated in four treatment groups: T1) CZB medium + bovine serum albumin (BSA) on sheep oviductal monolayer (SOM), T2) CZB + polyvinyl alcohol (PVA) + SOM, T3) CZB + PVA + SOM supplemented with inositol (I) and serine (S), T4) TCM 199 + 10% fetal calf serum + SOM. Standard culture conditions were 2 ml of medium in 35 mm Petri dishes, under 5% CO2 in air at 39 degrees C. The percentages of morulae and blastocysts were recorded after 4 and 7 days of culture. After 4 days of culture there was no significant difference (P > 0.05) in the percentage of morulae between embryos cultured in T1 (86%), T2 (85%), T3 (88.8%), and T4 (87.5%). After 7 days the percentages of blastocysts were T1 (70%), T2 (50%), T3 (55.5%) and T4 (46.8%). These data suggest that a protein-free medium, CZB + PVA and CZB + PVA + I + S, can support ovine preimplantation embryo development in vitro; however CZB medium supplemented with BSA enhances development to blastocyst.     

昆明白小鼠1细胞胚胎体外培养系统的研究

研究发现在有或者没有磷酸盐的条件下,葡萄糖均抑制昆明白小鼠ｌ－４细胞期胚胎的体外发 育。在不含葡萄糖和磷酸盐的ＨＥＣＭ－ｌ中,桑椹率为４０．０５％（７４／１６８）,而对照Ｇ－ＨＥＣＭ－１仅为 ８．１４％（７／８６）;不含葡萄糖含有磷酸盐的ＣＺＢ中,桑椹率为６７．１１％（９３／１５２）,而对照ＴＡＬＰ仅 ６· ６７％（６／９０）。用不含葡萄糖而含有１． ０ｍｍｏ１／Ｌ谷氨酸肢和０． １１ｍｍｏｌ／Ｌ ＥＤＴＡ的ＣＺＢ液,与兔输 卵管上皮单层培养细胞（ＲＯＥＣ）协同培养小鼠１细胞胚,７３．３３％（１１０／１５０）胚胎发育至桑椹胚, 但没有观察到囊胚形成、用上述ＣＺＨＲＯＥＣ系统培养小鼠１细胞胚４８小时（３－４细胞）,再移入 ＴＣＭ１９９＋１０％ＦＣＳ＋ＲＯＥＣ系统,有７６．７４％（６７／８６）胚胎发育至桑椹胚,９６／小时后,４０．７０％ （３５／８６）发育至囊胚。     

The Growth of Acer pseudoplatanus Cells in a Synthetic Liquid Medium: Response to the Carbohydrate, Nitrogenous and Growth Hormone Constituents

A synthetic culture medium which supports a high level of growth of a scrially propagated cell suspension culture of Acer pseudoplatanus is described. The sucrose of this medium can be effectively replaced by glucose or fructose or a mixture of glucose and fructose or galactose or maltose or soluble starch. When the carbohydrate is glucose or fructose no other sugars appear in the culture medium in significant amounts. Glucose is absorbed in greater quantity than fructose from an equimolar mixture of these sugars. When sucrose is supplied both glucose and fructose appear in the medium. Glucose appears in maltose medium, and maltose and glucose in soluble starch medium. Under the standard conditions of culture, media containing 2 % sucrose or 2 % glucose become depleted of sugar before the 25th day of incubation. Enhanced yield of the cultures can be obtained by raising the initial sucrose concentration to 6 %. – A supply of nitrate is essential for maximum yield and healthy growth. Growth, in the presence of nitrate, is significantly enhanced by a supply of urea. Addition of casein hydrolysate or of a mixture of amino acids enhances growth in the presence of nitrate and urea and particularly when nitrate is omitted. – When kinetin is omitted or incorporated at the standard level (0.25 mg/I), 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/l is essential for continuation of growth at a high level. It cannot be replaced by indol-3yl-acetic acid (IAA). 1-naphthaleneacetic acid (NAA) at 10 mg/l permits of a low level of growth with abnormal aggregation. When the level of kinetin is raised to 10 mg/l a high level of growth occurs in the absence of added auxin but the cultures become brown and tend to show increasing aggregation on subculture.     

Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose

The failure of hamster 2-cell embryos to develop in vitro (2-cell block) was examined with experiments in which concentrations of glucose and phosphate in the culture medium were varied. Embryos were cultured in a protein-free modified Tyrode's solution that normally contains 5.0 mM glucose and 0.35 mM sodium dihydrogen phosphate. In the presence of 0.35 mM phosphate but without glucose, 23% of 2-cell embryos reached the 4-cell stage or further after culture for 1 day and 27% after 2 days. Glucose inhibited embryo development even at 0.1 mM (4% development to greater than or equal to 4-cells after culture for 2 days); there was no dose-related inhibition above this glucose concentration. In a second experiment, phosphate levels were varied in the absence of glucose. Phosphate was highly inhibitory to development, with 97% of 2-cell embryos reaching the 4-cell stage or further after culture for 1 day in the absence of phosphate compared to 9-21% in the presence of 0.1-1.05 mM phosphate. After culture for 2 days, 26% of embryos reached the 8-cell stage or further when phosphate was absent compared to 0% development to 8-cells with 0.1 mM phosphate or higher. In a factorial experiment, phosphate blocked development when glucose was present or absent, whereas glucose did not block embryo development in the absence of phosphate. However, 2-deoxyglucose (a non-metabolizable analogue of glucose) inhibited embryo development in the absence of phosphate. These data show that the in vitro block to development of hamster 2-cell embryos is caused at least in part by glucose and/or phosphate. Deletion of these compounds from the culture medium eliminates the 2-cell block to development in virtually all embryos, and approximately 25-75% of embryos develop to the 8-cell or morula stages in vitro. The observations provide a possible explanation for the 2-cell and 4-cell blocks that occur in conventional culture media: stimulation of glycolysis by glucose and/or phosphate may result in inefficient adenosine triphosphate (ATP) production. The data indicate marked dissimilarities in the regulation of in vitro development of early cleavage stage hamster embryos compared with embryos of inbred mice, since the latter have an inactive glycolytic pathway prior to the 8-cell stage of development and will grow from 1-cell to blastocyst with both phosphate and glucose in the culture medium.     

In vitro development of ovine embryos in CZB medium

One- to four-cell embryos were collected from multiparous crossbred ewes and were cultured in vitro for 120 hours in CZB medium. A 2x2 factorial treatment arrangement was used to examine the effects of glucose and ethylenediaminetetraacetic acid (EDTA) on in vitro embryo development. The embryos were examined every 12 hours, and all of the embryos were stained with a DNA-specific fluorochrome after the 120-hour evaluation to enable the counting of cell nuclei. Embryo development was analyzed for cleavage beyond 16 cells as well as for cleavage to at least the compact morula stage based upon both the 120-hour morphological evaluation and nuclear counts. Forty-eight percent of the embryos passed through the in vitro developmental block (i.e., cleaved beyond 16 cells), and 26% developed to 30 or more cells. Neither EDTA nor glucose affected in vitro embryo development based on the nuclear counts.     

Characterization of mutations that overcome the toxic effect of glucose on phosphoglucose isomerase less strains of Saccharomyces cerevisiae

Abstract Glucose inhibits growth of yeast phosphoglucose isomerase mutants in permissive media. Mutants insensitive to this effect were isolated by selection on media containing 2% fructose + 2% glucose. A nuclear, monogenic, recessive mutation named rgl was responsible for this phenotype. The mutants isolated belonged to two complementation groups and have been termed rgl1 and rgl2 . When the double mutants were grown on fructose, fermentation of fructose or glucose was similar to that of the parental pgi strain but was not measurable when grown on fructose + glucose. Under these conditions, respiration of glucose and to a lesser extent of fructose was enhanced. The double mutants pgi rgl did not grow on fructose + glucose in the presence of antimycin A or ethidium bromide and their cytochrome oxidase was no longer sensitive to glucose repression. The results are interpreted as an indication that in the double mutants the glucose may be channeled through the pentose phosphate pathway to respiration.     

Synergism of glucose and fructose in net glycogen synthesis in perfused rat livers

Synergism of glucose and fructose in net glycogen synthesis was studied in perfused livers from 24-h fasted rats. With either glucose or fructose alone, net glycogen deposition did not occur (p greater than 0.10 for each), whereas the addition of both together resulted in significant glycogen accumulation (net glycogen accumulation was 0.21 +/- 0.03 mumol of glucose/g of liver/min at 2 mM fructose and 30 mM glucose, p less than 0.001). To better understand this synergism, intermediary substrate levels were compared at steady state with various glucose levels in the absence and in the presence of 2 mM fructose. Independent of fructose, hepatic glucose and glucose 6-phosphate increased proportionally when glucose level in the medium was raised (r = 0.86, p less than 0.001). Unlike glucose 6-phosphate, UDP-glucose did not consistently increase with glucose (p greater than 0.10); in fact, there was a small decrease at a very high glucose level (30 mM), a result consistent with the well-established activation of glycogen synthase by glucose. With elevated glucose, the level of glucose 6-phosphate was strongly correlated with glycogen content (r = 0.71, p less than 0.01, slope = 32). Adding fructose increased the "efficiency" of glucose 6-phosphate to glycogen conversion: the effect of a given increment in glucose 6-phosphate upon glycogen accumulation was increased 2.6-fold (r = 0.73, p less than 0.01, slope = 86). A kinetic modeling approach was used to investigate the mechanisms by which fructose synergized glycogen accumulation when glucose was elevated. Based on steady-state hepatic substrate levels, net hepatic glucose output, and net glycogen synthesis rate, the model estimated the rate constants of major enzymes and individual fluxes in the glycogen metabolic pathway. Modeling analysis is consistent with the following scenario: glycogen synthase is activated by glucose, whereas glucose-6-phosphatase was inhibited. In addition, the model supports the hypothesis that fructose synergizes net glycogen accumulation due to suppression of phosphorylase. Overall, our analysis suggests that glucose enhances the metabolic flux to glycogen by inducing a build up of glucose 6-phosphate via combined effects of mass action and glucose-6-phosphatase inhibition and activating glycogen synthase and that fructose enhances glycogen accumulation by retaining glycogen via phosphorylase inhibition.