Synthesis and degradation of phosphoenolpyruvate carboxylase in rat liver and adipose tissue. Changes during a starvation-re-feeding cycle

A specific antibody against liver cytosol phosphoenolpyruvate carboxylase (EC 4.1.1.32) was used to isolate the enzyme from liver and adipose tissue. With this technique we have shown that phosphoenolpyruvate carboxylase synthesis in starved rats accounts for 3% of the total synthesis of cytosol protein in each tissue. Re-feeding starved animals decreases this relative rate of phosphoenolpyruvate carboxylase synthesis to 0.2% and 1% respectively in liver and adipose tissue, and the activity of the enzyme in each tissue is decreased to 25% of the starvation value. An additional starvation period is accompanied by an increased rate of enzyme synthesis, but the response to starvation is considerably slower than that caused by re-feeding. The degradation rate of phosphoenolpyruvate carboxylase is also subject to regulation. Thus re-feeding starved animals decreases the half-life of the enzyme in liver from 13h to 5.2h, but the rapid rate of degradation is maintained at least during the first 20h of subsequent starvation. Only slight changes in the degradation rate of phosphoenolpyruvate carboxylase are found in adipose tissue. We conclude that the large alterations in the rate of enzyme synthesis during a starvation–re-feeding cycle are the major cause of fluctuations in activity.     

Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans

We report that four putative germline RNA helicases, GLHs, are components of the germline-specific P granules in Caenorhabditis elegans. GLH-3 and GLH-4, newly discovered, belong to a multi-gene glh family. Although GLHs are homologous to Drosophila VASA, a polar granule component necessary for oogenesis and embryonic pattern formation, the GLHs are distinguished by containing multiple CCHC zinc fingers. RNA-mediated interference (RNAi) reveals the GLHs are critical for oogenesis. By RNAi at 20 degrees C, when either loss of GLH-1 or GLH-4 alone has no effect, loss of both GLH-1 and GLH-4 results in 97% sterility in the glh-1/4(RNAi) offspring of injected hermaphrodites. glh-1/4(RNAi) germlines are under-proliferated and are without oocytes. glh-1/4(RNAi) animals produce sperm; however, spermatogenesis is delayed and the sperm are defective. P granules are still present in glh-1/4(RNAi) sterile worms as revealed with antibodies against the remaining GLH-2 and GLH-3 proteins, indicating the GLHs function independently in P granule assembly. These studies reveal that C.elegans can use GLH-1 or GLH-4 to promote germline development.     

Gld-1, a Tumor Suppressor Gene Required for Oocyte Development in Caenorhabditis Elegans

We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1(null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1(null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells.     

Eggs over easy: cell death in the Drosophila ovary

Programmed cell death is the most common fate of female germ cells in Drosophila and many animals. In Drosophila, oocytes form in individual egg chambers that are supported by germline nurse cells and surrounded by somatic follicle cells. As oogenesis proceeds, 15 nurse cells die for every oocyte that is produced. In addition to this developmentally regulated cell death, groups of germ cells or entire egg chambers may be induced to undergo apoptosis in response to starvation or other insults. Recent findings suggest that these different types of cell death involve distinct genetic pathways. This review focuses on progress towards elucidating the molecular mechanisms acting during programmed cell death in Drosophila oogenesis.     

Citrate and the conversion of carbohydrate in fat. The activities of citrate-cleavage enzyme and acetate thiokinase in livers of starved and re-fed rats

1. The activity of citrate-cleavage enzyme varies in accordance with the nutritional state of the animal. It is suppressed on starvation and restored on re-feeding after starvation. 2. The increase in enzyme activity that occurs on re-feeding starved animals depends on the diet. It is largest on diets high in carbohydrate and low in fat, and smallest on diets high in fat. Intermediate increases are obtained with balanced diets. 3. The ratio of activities of citrate-cleavage enzyme to acetate thiokinase varies from 2·5 for animals maintained on a balanced diet to 20 for animals re-fed with a diet high in carbohydrate. 4. The changes in activity of citrate-cleavage enzyme correlate with changes in the rate of fatty acid synthesis and provide evidence for the involvement of the citrate-cleavage reaction in fatty acid synthesis.     

Selective changes in the protein-turnover rates and nature of growth induced in trout liver by long-term starvation followed by re-feeding

We report upon the effects of a cycle of long-term starvation followed by re-feeding on the liver-protein turnover rates and nature of protein growth in the rainbow trout (Oncorhynchus mykiss). We determined the protein-turnover rate and its relationship with the nucleic-acid concentrations in the livers of juvenile trout starved for 70 days and then re-fed for 9 days. During starvation the total hepatic-protein and RNA contents decreased significantly and the absolute protein-synthesis rate (A_S) also fell, whilst the fractional protein-synthesis rate (K_S) remained unchanged and the fractional protein-degradation rate (K_D) increased significantly. Total DNA content, an indicator of hyperplasia, and the protein:DNA ratio, an indicator of hypertrophy, both fell considerably. After re-feeding for 9 days the protein-accumulation rates (K_G, A_G) rose sharply, as did K_S, A_S, K_D, protein-synthesis efficiency (K_RNA) and the protein-synthesis rate/DNA unit (K_DNA). The total hepatic protein and RNA contents increased but still remained below the control values. The protein:DNA and RNA:DNA ratios increased significantly compared to starved fish. These changes demonstrate the high response capacity of the protein-turnover rates in trout liver upon re-feeding after long-term starvation. Upon re-feeding hypertrophic growth increased considerably whilst hyperplasia remained at starvation levels.     

Deadlock, a novel protein of Drosophila, is required for germline maintenance, fusome morphogenesis and axial patterning in oogenesis and associates with centrosomes in the early embryo

The deadlock gene is required for a number of key developmental events in Drosophila oogenesis. Females homozygous for mutations in the deadlock gene lay few eggs and those exhibit severe patterning defects along both the anterior-posterior and dorsal-ventral axis. In this study, we analyzed eggs and ovaries from deadlock mutants and determined that deadlock is required for germline maintenance, stability of mitotic spindles, localization of patterning determinants, oocyte growth and fusome biogenesis in males and females. Deadlock encodes a novel protein which colocalizes with the oocyte nucleus at midstages of oogenesis and with the centrosomes of early embryos. Our genetic and immunohistological experiments point to a role for Deadlock in microtubule function during oogenesis.     

家兔供体细胞的发育周期与重构胚发育的关系

采用血清饥饿法处理体外培养的兔子胎儿成纤维细胞,并将其作为供体细胞移入去核卵母细胞内构建重构胚胎。检查供体细胞的细胞周期对重构胚的融合率、分裂率和着床率的影响。实验结果表明：培养基中血清含量在0．5％的情况下,G0／G1期的细胞比例由正常培养条件下(培乔液中含有10％FCS)的73．2％明显地增加到86％以上。饥饿1～3天的细胞作为供体细胞构建重构胚时,可明显提高重构胚的融合率,但是不同的饥饿时间其融合率并无显著的差异。饥饿处理可明显增加重构胚的分裂率,以饥饿处理3天为最佳。     

Regulation of the sperm‐to‐oocyte transition in Caenorhabditis briggsae hermaphrodites by the Cbr‐met‐2 and Cbr‐fem‐3 genes

In Caenorhabditis briggsae hermaphrodites, spermatogenesis begins in the L4 larval stage and persists into early adulthood. Oogenesis begins after spermatogenesis; the sperm‐to‐oocyte transition is irreversible. The timing of this transition is believed to have evolved in response to selection to maximize the intrinsic growth rate. Sperm‐to‐oocyte transitions occurred early in Cbr‐met‐2 and Cbr‐fem‐3 mutants. These early transitions resulted in reduced brood sizes, but had little or no impact on the intrinsic growth rate. In Cbr‐met‐2; Cbr‐fem‐3 doubly mutant hermaphrodites, the transition to oogenesis occurred even earlier and brood size was further reduced, indicating that Cbr‐met‐2 and Cbr‐fem‐3 regulate the sperm‐to‐oocyte transition through separate pathways. Mutations in Cbr‐met‐2 also resulted in an increase in the frequency of males in mutant populations. These increased male frequencies were not caused by increased rates of X nondisjunction during oogenesis in mutant hermaphrodites. Rather, increases in the rates of outcrossing in mutant populations likely were an indirect effect of reduced brood sizes derived from self‐fertilization. Based on these observations, it is possible that the timing of the sperm‐to‐oocyte transition in C. briggsae evolved in response to sexual selection on hermaphrodites to limit rates of outcrossing. Mutations in the orthologous Caenorhabditis elegans gene, Cel‐met‐2, did not impact the timing of the sperm‐to‐oocyte transition, consistent with the independent evolution of hermaphroditic reproduction in these species. Although brood sizes were reduced in Cel‐met‐2 mutant strains, increased male frequencies were not observed. Cbr‐ and Cel‐met‐2 mutations also differed in terms of germline mortality, observed in C. elegans, but not in C. briggsae.     

Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.     

The role of food availability in regulating reproductive development in female golden perch

At a time of the year when female golden perch Macquaria ambigua are not normally reproductively active, they were either fed daily to satiety (Fed), starved for 150 days (S150), or starved for 150 days then fed to satiety for 30 or 60 days (S150/F30 or S150/F60). Fish showed rapid growth and increased food conversion efficiency upon re-feeding relative to Fed animals. The hepatosomatic indices were not significantly different between Fed, S150/F30 and S150/F60 groups, but were significantly reduced in S150 animals. The gonadosomatic indices ( I _G) for both Fed and S150 animals were not significantly different. However, the I _G values for S150/F30 and S150/F60 animals of 6·74±1·22 and 7·84±1·12 were significantly elevated relative to Fed animals and approach those described for wild mature M. ambigua . Oocyte development in Fed and S150 animals did not proceed past the cortical alveoli or perinucleolar stages, respectively, but oocytes in both S150/F30 and S150/F60 animals had undergone vitellogenesis and were close to being mature. The concentration of oestradiol and testosterone in the plasma of S150/F30 and S150/F60 animals increased in accordance with the proposed role of these hormones in teleost reproductive cycles. The reproductive response of M. ambigua to starvation and re-feeding is well suited to reproductive success in temperate Australian rivers where food availability is unpredictable.     

西伯利亚鲟在高温下饥饿后的补偿生长

在高温(29±1)℃下将西伯利亚鲟幼鱼(21.61±0.03)g饥饿0(对照)、6、12和18d后恢复摄食3周, 研究摄食、生长和鱼体组成的变化。结果表明, 经过不同程度饥饿的鱼体重均显著低于对照组(P0.05), 而饥饿18d(S18组)的鱼体重显著低于对照(P0.05)。在饥饿过程中,鱼体脂肪含量和肝脏肝糖原含量下降的同时, 各饥饿组的灰分含量上升, 但仅S18组与对照差异显著(P0.05)。结果表明, 西伯利亚鲟在高温下表现出完全补偿现象, 且是通过同时提高摄食率和饲料效率来实现补偿生长的, 因此在夏季高温时对鲟鱼进行一段时间适度的饥饿可以在不影响生长和体成分的前提下节约饲料成本, 减少因过量投饵而引起的环境污染。       

The Caenorhabditis elegans homologue of deleted in azoospermia is involved in the sperm/oocyte switch

The Deleted in Azoospermia (DAZ) gene family encodes putative translational activators that are required for meiosis and other aspects of gametogenesis in animals. The single Caenorhabditis elegans homologue of DAZ, daz-1, is an essential factor for female meiosis. Here, we show that daz-1 is important for the switch from spermatogenesis to oogenesis (the sperm/oocyte switch), which is an essential step for the hermaphrodite germline to produce oocytes. RNA interference of the daz-1 orthologue in a related nematode, Caenorhabditis briggsae, resulted in a complete loss of the sperm/oocyte switch. The C. elegans hermaphrodite deficient in daz-1 also revealed a failure in the sperm/oocyte switch if the genetic background was conditional masculinization of germline. DAZ-1 could bind specifically to mRNAs encoding the FBF proteins, which are translational regulators for the sperm/oocyte switch and germ stem cell proliferation. Expression of the FBF proteins seemed to be lowered in the daz-1 mutant at the stage for the sperm/oocyte switch. Conversely, a mutation in gld-3, a gene that functionally counteracts FBF, could partially restore oogenesis in the daz-1 mutant. Together, we propose that daz-1 plays a role upstream of the pathway for germ cell sex determination.     

Oskar organizes the germ plasm and directs localization of the posterior determinant nanos

Oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. We have cloned oskar and show that oskar RNA is localized to the posterior pole of the oocyte when germ plasm forms. This polar distribution of oskar RNA is established during oogenesis in three phases: accumulation in the oocyte, transport toward the posterior, and finally maintenance at the posterior pole of the oocyte. The colocalization of oskar and nanos in wild-type and bicaudal embryos suggests that oskar directs localization of the posterior determinant nanos. We propose that the pole plasm is assembled stepwise and that continued interaction among its components is required for germ cell determination.     

Reversible response of protein localization and microtubule organization to nutrient stress during Drosophila early oogenesis

The maturation of animal oocytes is highly sensitive to nutrient availability. During Drosophila oogenesis, a prominent metabolic checkpoint occurs at the onset of yolk uptake (vitellogenesis): under nutrient stress, egg chambers degenerate by apoptosis. To investigate additional responses to nutrient deprivation, we studied the intercellular transport of cytoplasmic components between nurse cells and the oocyte during previtellogenic stages. Using GFP protein-traps, we showed that Ypsilon Schachtel (Yps), a putative RNA binding protein, moved into the oocyte by both microtubule (MT)-dependent and -independent mechanisms, and was retained in the oocyte in a MT-dependent manner. These data suggest that oocyte enrichment is accomplished by a combination of MT-dependent polarized transport and MT-independent flow coupled with MT-dependent trapping within the oocyte. Under nutrient stress, Yps and other components of the oskar ribonucleoprotein complex accumulated in large processing bodies in nurse cells, accompanied by MT reorganization. This response was detected as early as 2 h after starvation, suggesting that young egg chambers rapidly respond to nutrient stress. Moreover, both Yps aggregation and MT reorganization were reversed with re-feeding of females or the addition of exogenous insulin to cultured egg chambers. Our results suggest that egg chambers rapidly mount a stress response by altering intercellular transport upon starvation. This response implies a mechanism for preserving young egg chambers so that egg production can rapidly resume when nutrient availability improves.     

Effects of starvation and feeding upon corpus allatum activity and oöcyte growth in adult female Periplaneta americana

Direct radiochemical determinations of juvenile hormone (JH) biosynthesis by corpora allata (CA) isolated from starved and re-fed Periplanteta americana have been employed to elucidate the humoral mechanisms involved in the modulation of reproductive activity in response to food availability. When starvation was initiated in mature adult females at the time of formation of an oötheca the next oötheca was normally deposited 5 to 6 days later, a delay of 2–3 days, and a third oötheca was formed by only 50% of starved females. The terminal oöcytes in the remaining females were either resorbed or maintained in an arrested state. Ovarian development had effectively ceased after 2 weeks of starvation but recommenced within 3 days of re-feeding. The CA of most starved females exhibited 2 activity cycles following food withdrawal. The first peak occurred on day 1 of the starvation period and was coincident with the timing for fed controls. The second peak was delayed by about 2 days and the activity of the CA then declined to the extent that glands from animals starved for more than 11 days were completely inactive. Feeding, after 10 or 16 days starvation, resulted in a resumption of CA activity which was detectable in some animals within 24 hr, and very high rates of JH biosynthesis were found 4 or 5 days later. The results suggest that P. americana can readily and efficiently modulate egg production in response to food supply, and that control is effected through alterations in JH production by the CA. The use of farnesenic acid as a biochemical probe indicates that CA inactivity after long periods of starvation does not arise because malnutrition has caused complete metabolic shut-down in the glands, and that JH biosynthesis is basically modulated at a control point prior to the last two enzymic stages in the pathway.     

Comparison of tissue pyruvate dehydrogenase activities on re-feeding rats fed ad libitum or meal-fed rats with a chow-diet meal.

Meal-fed rats and rats fed ad libitum had similar rates of hepatic glycogenesis at 60 min after the initiation of re-feeding a chow meal after 22 h starvation, but hepatic PDHa (active form of pyruvate dehydrogenase) activities were 4-fold higher in the meal-fed group. In heart, PDHa activities were 3-fold higher before re-feeding and 2-fold higher after re-feeding in the meal-fed group compared with the group fed ad lib. The blood metabolite profile suggested diminished fat oxidation in starved meal-fed rats and accelerated flux through PDH in meal-fed re-fed rats compared with the group fed ad lib.     

Direction of carbon flux in starvation and after refeeding: in vitro and in vivo effects of 3-mercaptopicolinate

3-Mercaptopicolinate (3-MPA) is a specific inhibitor of phosphoenolpyruvate carboxykinase (PEP CK). In vivo the hypoglycaemic action of 3-MPA in 24 h-starved rats was abolished on intragastric glucose refeeding. Nonetheless, 3-MPA decreased hepatic glycogen content and rate of synthesis in starved animals re-fed glucose. The inference is that on re-feeding after starvation hepatic glycogen is synthesised mainly de novo via glyconeogenesis involving PEP CK. 3-MPA increased hepatic lipogenesis in water- and glucose-fed normal and diabetic rats. This increase is presumed to result from inhibition of PEP CK and consequent diversion of pyruvate from gluconeogenesis to lipogenesis. In contrast, 3-MPA inhibited brown-fat lipogenesis in water- and glucose-fed rats.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.