MicroRNA-29a mediates the impairment of intestinal epithelial integrity induced by intrauterine growth restriction in pig.

An important characteristic of intrauterine growth restricted (IUGR) neonate is the impaired intestinal barrier function. With the use of a pig model, this study was conducted to identify the responsible microRNA (miRNA) for the intestinal damage in IUGR neonates through comparing the miRNA profile of IUGR and normal porcine neonates and to investigate the regulation mechanism. Compared with the normal ones, we identified 83 upregulated and 76 downregulated miRNAs in the jejunum of IUGR pigs. Notably, IUGR is associated with profoundly increasesd miR-29 family and decreased expression of extracellular matrix (ECM) and tight junction (TJ) proteins in the jejunum. Furthermore, in vitro study using theporcine intestinal epithelial cell line (IPEC-1) showed that inhibition of miR-29a expression could improve the monolayer integrity by increasing cell proliferation and transepithelial resistance. Also, overexpression/inhibition of miR-29a in IPEC-1 cells can suppress/increase the expression of integrin-ß1, collagen I, collagen IV, fibronectin, and claudin 1, both at transcriptional and translational levels. Subsequent luciferase reporter assay confirmed a direct interaction between miR-29a and the 3'-untranslated regions of these genes. In conclusion, this study reveals that IUGR-impaired intestinal barrier function is associated with downregulated ECM and TJ protein expression mediated by the upregulation of miR-29a.NEW & NOTEWORTHY Intrauterine growth restricted (IUGR) remains a major problem for both human health and animal production due to its association with high rates of preweaning morbidity and mortality. We have identified the abnormal expression of microRNA-29a (miR-29a) in the small intestine of IUGR neonates, as well as its targets and mechanisms. These results provide new information about biological characteristics of IUGR-affected intestinal dysfunction and can lead to the development of potentially solution for preventing and treating IUGR in the future.

L-Glutamine deprivation induces autophagy and alters the mTOR and MAPK signaling pathways in porcine intestinal epithelial cells.

L-Glutamine (Gln) is an essential amino acid for intestinal growth and integrity. However, the underlying molecular mechanisms are not fully known. In the present study, porcine intestinal epithelial cells (IPEC-1) were used to test the hypothesis that autophagy is induced by Gln deprivation and inhibited by Gln supplementation. After a 2-day period of growth in normal medium, IPEC-1 cells were transferred to a Gln-free custom-made DMEM. Cell numbers, the distribution of autophagosomes, the abundance of the protein for an autophagy marker LC3B, as well as abundances of the mTOR and MAPK proteins during an 8-h period were determined. Furthermore, the rescue effect of 5 mM Gln was evaluated. Our results showed that Gln deprivation reduced the cell number, while enhancing the accumulation of autophagosomes and the expression of LC3B-II in IPEC-1 cells within 8 h. The concentrations of Glu, Asp, Cit, Arg, Leu, Ile, Val, Ala, ß-Ala, Orn, Phe, Met and Ser in the culture medium were altered by Gln deprivation. Further analysis revealed that Gln deficiency inactivated, but Gln supplementation activated, the mTOR and MAPK/ERK signaling pathways. Collectively, our findings support the notion that Gln deficiency induces autophagy and disturbs amino acid metabolism in intestinal epithelial cells, as well as attenuated their mTOR and MAPK/ERK signaling pathways to inhibit protein synthesis and cell proliferation.

Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity.

The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.

Human SIRT5 variants with reduced stability and activity do not cause neuropathology in mice.

SIRT5 is a sirtuin deacylase that removes negatively charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal conditions, the phenotypes of SIRT5 deficiency are quite subtle. Here, we identify two homozygous SIRT5 variants in patients suspected to have mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generated a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology, or other gross phenotypes. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, but are likely not by themselves the primary pathogenic cause of the neuropathology observed in the patients.

SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.

OCRbayes: A Bayesian hierarchical modeling framework for Seahorse extracellular flux oxygen consumption rate data analysis.

BACKGROUND: Mitochondrial dysfunction is involved in many complex diseases. Efficient and accurate evaluation of mitochondrial functionality is crucial for understanding pathology as well as facilitating novel therapeutic developments. As a popular platform, Seahorse extracellular flux (XF) analyzer is widely used for measuring mitochondrial oxygen consumption rate (OCR) in living cells. A hidden feature of Seahorse XF OCR data is that it has a complex data structure, caused by nesting and crossing between measurement cycles, wells and plates. Surprisingly, statistical analysis of Seahorse XF data has not received sufficient attention, and current methods completely ignore the complex data structure, impairing the robustness of statistical inference. RESULTS: To rigorously incorporate the complex structure into data analysis, here we developed a Bayesian hierarchical modeling framework, OCRbayes, and demonstrated its applicability based on analysis of published data sets. CONCLUSIONS: We showed that OCRbayes can analyze Seahorse XF OCR experimental data derived from either single or multiple plates. Moreover, OCRbayes has potential to be used for diagnosing patients with mitochondrial diseases.

Glucosamine Supplementation in Premating Drinking Water Improves Within-Litter Birth Weight Uniformity of Rats Partly through Modulating Hormone Metabolism and Genes Involved in Implantation.

Within-litter birth weight variation in multiparous animals has become a big issue due to high incidence of low birth weight neonates, which gives rise to high preweaning mortality and morbidity. Foetus with various birth weights is the outcome of diverse embryos competence which is affected by oocyte quality. Glucosamine (GlcN) has been reported to be involved in oocyte maturation; however, its effect on pregnant outcomes remains unknown. The present study was conducted to investigate the effects of premating GlcN supplementation via drinking water on within-litter birth weight variation and its underlying mechanism. Fifty eight Sprague-Dawley female rats were randomly assigned to one of two groups with normal drinking water or drinking water supplemented with 0.5 mM GlcN from six to eight weeks old. Variation of within-litter birth weight in the GlcN group was 5.55%, significantly lower compared with 8.17% in the control group. Birth weight was significantly increased in the GlcN group (2.27 ± 0.06) compared with the control group (2.08 ± 0.04). Both absolute and relative weights of the ovary at the end of GlcN treatment were higher in the GlcN group than in the control group (P < 0.05). In the GlcN group, there were more successfully implanted blastocysts (13.38 ± 0.63 and 15.75 ± 0.59 in the control and treatment group, respectively) with more uniform distribution along the two uterine horns compared with the control group. Besides, gene expressions of Alk3 and Bmp2 were increased in the implantation sites, while IGF-1 and Mucin-1 were decreased significantly in rats administrated with GlcN. Maternal progesterone, estradiol, and IGF-1 concentrations on D 19.5 were significantly increased, while insulin and total cholesterol levels were significantly decreased in contrast with control dams. In summary, the administration of 0.5 mM GlcN solution before mating reduced within-litter birth weight variation, accompanied with increased fetal weight. Further investigation indicated that the improved outcome of pregnancy results at least partly from the increased ovary weights of the rats, the homogeneous embryo developmental competence, the enhanced receptivity of the uterine environment, and the adjusted maternal hormone levels.

An optimized desuccinylase activity assay reveals a difference in desuccinylation activity between proliferative and differentiated cells.

Succinylation is a novel post-translational modification identified on many proteins and is involved in multiple biological processes. Succinylation levels are dynamically regulated, balanced by succinylation and desuccinylation processes, and are closely connected to metabolic state in vivo. Sirtuins have been shown to possess NAD+-dependent desuccinylation activity in vitro and in vivo, among which the desuccinylation activity of SIRT5 is most extensively studied. Our understanding of the response of succinylation levels to different metabolic conditions, is hampered by the lack of a fast NAD+-dependent desuccinylation assay in a physiological context. In the present study, we therefore optimized and validated a fluorescence-based assay for measuring NAD+-dependent desuccinylation activity in cell lysates. Our results demonstrated that shorter and stricter reaction time was critical to approach the initial rate of NAD+-dependent desuccinylation activity in crude cell lysate systems, as compared to the desuccinylation reaction of purified His-SIRT5. Analysis of desuccinylation activity in SIRT5 knockout HEK293T cells confirmed the relevance of SIRT5 in cellular desuccinylation activity, as well as the presence of other NAD+-dependent desuccinylase activities. In addition, we were able to analyse desuccinylation and deacetylation activity in multiple cell lines using this assay. We showed a remarkably higher desuccinylase activity, but not deacetylase activity, in proliferative cultured muscle and adipose cells in comparison with their differentiated counterparts. Our results reveal an alteration in NAD+-dependent desuccinylation activity under different metabolic states.

Within-litter variation in birth weight: impact of nutritional status in the sow.

Accompanying the beneficial improvement in litter size from genetic selection for high-prolificacy sows, within-litter variation in birth weight has increased with detrimental effects on post-natal growth and survival due to an increase in the proportion of piglets with low birth-weight. Causes of within-litter variation in birth weight include breed characteristics that affect uterine space, ovulation rate, degree of maturation of oocytes, duration of time required for ovulation, interval between ovulation and fertilization, uterine capacity for implantation and placentation, size and efficiency of placental transport of nutrients, communication between conceptus/fetus and maternal systems, as well as nutritional status and environmental influences during gestation. Because these factors contribute to within-litter variation in birth weight, nutritional status of the sow to improve fetal-placental development must focus on the following three important stages in the reproductive cycle: pre-mating or weaning to estrus, early gestation and late gestation. The goal is to increase the homogeneity of development of oocytes and conceptuses, decrease variations in conceptus development during implantation and placentation, and improve birth weights of newborn piglets. Though some progress has been made in nutritional regulation of within-litter variation in the birth weight of piglets, additional studies, with a focus on and insights into molecular mechanisms of reproductive physiology from the aspects of maternal growth and offspring development, as well as their regulation by nutrients provided to the sow, are urgently needed.