CAPE (caffeic acid phenethyl ester) stimulates glucose uptake through AMPK (AMP-activated protein kinase) activation in skeletal muscle cells

Caffeic acid phenethyl ester (CAPE), a flavonoid-like compound, is one of the major components of honeybee propolis. In the present study, we investigated the metabolic effects of CAPE in skeletal muscle cells and found that CAPE stimulated glucose uptake in differentiated L6 rat myoblast cells and also activated AMPK (AMP-activated protein kinase). In addition, the inhibition of AMPK blocked CAPE-induced glucose uptake, and CAPE activated the Akt pathway in a PI3K (phosphoinositide 3-kinase)-dependent manner. Furthermore, CAPE enhanced both insulin-mediated Akt activation and glucose uptake. In summary, our results suggest that CAPE may have beneficial roles in glucose metabolism via stimulation of the AMPK pathway.     

Identifying and quantifying metabolites by scoring peaks of GC-MS data

Background

Metabolomics is one of most recent omics technologies. It has been applied on fields such as food science, nutrition, drug discovery and systems biology. For this, gas chromatography-mass spectrometry (GC-MS) has been largely applied and many computational tools have been developed to support the analysis of metabolomics data. Among them, AMDIS is perhaps the most used tool for identifying and quantifying metabolites. However, AMDIS generates a high number of false-positives and does not have an interface amenable for high-throughput data analysis. Although additional computational tools have been developed for processing AMDIS results and to perform normalisations and statistical analysis of metabolomics data, there is not yet a single free software or package able to reliably identify and quantify metabolites analysed by GC-MS.

Results

Here we introduce a new algorithm, PScore, able to score peaks according to their likelihood of representing metabolites defined in a mass spectral library. We implemented PScore in a R package called MetaBox and evaluated the applicability and potential of MetaBox by comparing its performance against AMDIS results when analysing volatile organic compounds (VOC) from standard mixtures of metabolites and from female and male mice faecal samples. MetaBox reported lower percentages of false positives and false negatives, and was able to report a higher number of potential biomarkers associated to the metabolism of female and male mice.

Conclusions

Identification and quantification of metabolites is among the most critical and time-consuming steps in GC-MS metabolome analysis. Here we present an algorithm implemented in a R package, which allows users to construct flexible pipelines and analyse metabolomics data in a high-throughput manner.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0374-2) contains supplementary material, which is available to authorized users.     

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.     

A novel tool for individual haplotype inference using mixed data

Background

In many studies, researchers may recruit samples consisting of independent trios and unrelated individuals. However, most of the currently available haplotype inference methods do not cope well with these kinds of mixed data sets.

Methods

We propose a general and simple methodology using a mixture of weighted multinomial (MIXMUL) approach that combines separate haplotype information from unrelated individuals and independent trios for haplotype inference to the individual level.

Results

The new MIXMUL procedure improves over existing methods in that it can accurately estimate haplotype frequencies from mixed data sets and output probable haplotype pairs in optimized reconstruction outcomes for all subjects that have contributed to estimation. Simulation results showed that this new MIXMUL procedure competes well with the EM-based method, i.e. FAMHAP, under a few assumed scenarios.

Conclusion

The results showed that MIXMUL can provide accurate estimates similar to those haplotype frequencies obtained from FAMHAP and output the probable haplotype pairs in the most optimal reconstruction outcome for all subjects that have contributed to estimation. If available data consist of combinations of unrelated individuals and independent trios, the MIXMUL procedure can be used to estimate the haplotype frequencies accurately and output the most likely reconstructed haplotype pairs of each subject in the estimation.     

Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.     

mTOR is required for asymmetric division through small GTPases in mouse oocytes

Mammalian target of rapamycin (mTOR) is central to the control of cell proliferation, growth, and survival in mammalian cells. Prolonged treatment with rapamycin inhibits mTOR complex 2 (mTORC2) activity, and both the mTORC1-mediated S6K1 and 4E-BP1/eIF4E pathways are essential for TORC2-mediated RhoA, Cdc42, and Rac1 expression during cell motility and F-actin reorganization. The functions of mTOR in the mouse oocyte remain unclear, however. The present study shows that rapamycin affects mTOR expression and cytoskeleton reorganization during meiotic maturation of mouse oocytes. mTOR mRNA was expressed in germinal vesicles (GV) until metaphase I (MI), and increased during metaphase II (MII). Immunostaining showed that mTOR localized around the spindle and in the cytoplasm of oocytes. Treatment of oocytes with rapamycin decreased mTOR at the RNA and protein level, and altered asymmetric division. Formation of the actin cap and the cortical granule-free domain were also disrupted after rapamycin treatment, indicating the failure of spindle migration. Injection of an anti-mTOR antibody yielded results consistent with those obtained for rapamycin treatment, further confirming the involvement of mTOR in oocyte polarity. Furthermore, rapamycin treatment reduced the mRNA expression of small GTPases (RhoA, Cdc42, and Rac1), which are crucial regulatory factors for cytoskeleton reorganization. Taken together, these results suggest that rapamycin inhibits spindle migration and asymmetric division during mouse oocyte maturation via mTOR-mediated small GTPase signaling pathways.     

Ecological cues, gestation length, and birth timing in African buffalo (Syncerus caffer)

We examined annual variation in the timing of conception andparturition in the African buffalo (Syncerus caffer) and thesynchrony of birth timing with resource cues, using 8 yearsof monthly birth, rainfall, and vegetation data, measured asNormalized Difference Vegetation Index (NDVI). Monthly birthshad the strongest significant correlations with NDVI and rainfalllevels 12 and 13 months in the past, respectively. In addition,the synchrony of current year births corresponds most stronglyto the synchrony of the previous year's NDVI distribution. Becausethe gestation period of buffalo has been estimated to be around11 months, these findings suggest that improved protein levels,occurring approximately a month after the first green flushof the wet season, are either a trigger for conception or conceptionhas evolved to be synchronous with correlated environmentalcues that ensure females enter a period of peak body conditionaround the time of conception and/or parturition. With a gestationperiod of approximately 340 days, parturition occurs to takeadvantage of the period when forage has its highest proteincontent. A comparative analysis of gestation periods withinthe subfamily Bovinae indicates that African buffalo have aprotracted gestation for their body size, which we suggest isan adaptation to their seasonal environment. We also found thatinterannual variation in the birth distribution suggests a degreeof plasticity in the date of conception, and variation in thenumber of calves born each year suggest further synchrony ata timescale longer than a single year.