AMPK Regulates DNA Methylation of PGC-1α and Myogenic Differentiation in Human Mesenchymal Stem Cells.

Adverse intrauterine environments can cause persistent changes in epigenetic profiles of stem cells, increasing susceptibility of the offspring to developing metabolic diseases later in life. Effective approaches to restore the epigenetic landscape and function of stem cells remain to be determined. In this study, we investigated the effects of pharmaceutical activation of AMP-activated protein kinase (AMPK), an essential regulator of energy metabolism, on mitochondrial programming of Wharton's Jelly mesenchymal stem cells (WJ-MSCs) from women with diabetes during pregnancy. Induction of myogenic differentiation of WJ-MSCs was associated with increased proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression and mitochondrial DNA (mtDNA) abundance. Inhibition of DNA methylation by 5 Azacytidine significantly increased PGC-1α expression and mtDNA abundance in WJ-MSCs, which were abolished by AMPK inhibitor Compound C (CC), suggesting an AMPK-dependent role of DNA demethylation in regulating mitochondrial biogenesis in WJ-MSCs. Furthermore, activation of AMPK in diabetic WJ-MSCs by AICAR or metformin decreased the level of PGC-1α promoter methylation and increased PGC-1α expression. Notably, decreased PGC-1α promoter methylation by transient treatment of AMPK activators persisted after myogenic differentiation. This was associated with enhanced myogenic differentiation capacity of human WJ-MSCs and increased mitochondrial function. Taken together, our findings revealed an important role for AMPK activators in epigenetic regulation of mitochondrial biogenesis and myogenesis in WJ-MSCs, which could lead to potential therapeutics for preventing fetal mitochondrial programming and long-term adverse outcome in offspring of women with diabetes during pregnancy.

miR-130b/301b Is a Negative Regulator of Beige Adipogenesis and Energy Metabolism In Vitro and In Vivo.

Thermogenic brown or beige adipocytes dissipate energy in the form of heat and thereby counteract obesity and related metabolic complications. The miRNA cluster miR-130b/301b is highly expressed in adipose tissues and has been implicated in metabolic diseases as a posttranscriptional regulator of mitochondrial biogenesis and lipid metabolism. We investigated the roles of miR-130b/301b in regulating beige adipogenesis in vivo and in vitro. miR-130b/301b declined in adipose progenitor cells during beige adipogenesis, while forced overexpression of miR-130b-3p or miR-301b-3p suppressed uncoupling protein 1 (UCP1) and mitochondrial respiration, suggesting that a decline in miR-130b-3p or miR-301b-3p is required for adipocyte precursors to develop the beige phenotype. Mechanistically, miR-130b/301b directly targeted AMP-activated protein kinase (AMPKα1) and suppressed peroxisome proliferator-activated receptor γ coactivator-1α (Pgc-1α), key regulators of brown adipogenesis and mitochondrial biogenesis. Mice lacking the miR-130b/301b miRNA cluster showed reduced visceral adiposity and less weight gain. miR-130b/301b null mice exhibited improved glucose tolerance, increased UCP1 and AMPK activation in subcutaneous fat (inguinal white adipose tissue [iWAT]), and increased response to cold-induced energy expenditure. Together, these data identify the miR-130b/301b cluster as a new regulator that suppresses beige adipogenesis involving PGC-1α and AMPK signaling in iWAT and is therefore a potential therapeutic target against obesity and related metabolic disorders.

Interrogating congenital heart defects with noninvasive fetal echocardiography in a mouse forward genetic screen.

BACKGROUND: Congenital heart disease (CHD) has a multifactorial pathogenesis, but a genetic contribution is indicated by heritability studies. To investigate the spectrum of CHD with a genetic pathogenesis, we conducted a forward genetic screen in inbred mice using fetal echocardiography to recover mutants with CHD. Mice are ideally suited for these studies given that they have the same four-chamber cardiac anatomy that is the substrate for CHD. METHODS AND RESULTS: Ethylnitrosourea mutagenized mice were ultrasound-interrogated by fetal echocardiography using a clinical ultrasound system, and fetuses suspected to have cardiac abnormalities were further interrogated with an ultrahigh-frequency ultrasound biomicroscopy. Scanning of 46 270 fetuses revealed 1722 with cardiac anomalies, with 27.9% dying prenatally. Most of the structural heart defects can be diagnosed using ultrasound biomicroscopy but not with the clinical ultrasound system. Confirmation with analysis by necropsy and histopathology showed excellent diagnostic capability of ultrasound biomicroscopy for most CHDs. Ventricular septal defect was the most common CHD observed, whereas outflow tract and atrioventricular septal defects were the most prevalent complex CHD. Cardiac/visceral organ situs defects were observed at surprisingly high incidence. The rarest CHD found was hypoplastic left heart syndrome, a phenotype never seen in mice previously. CONCLUSIONS: We developed a high-throughput, 2-tier ultrasound phenotyping strategy for efficient recovery of even rare CHD phenotypes, including the first mouse models of hypoplastic left heart syndrome. Our findings support a genetic pathogenesis for a wide spectrum of CHDs and suggest that the disruption of left-right patterning may play an important role in CHD.

Anti-inflammatory activity of Crinum asiaticum Linne var. japonicum extract and its application as a cosmeceutical ingredient.

Crinum asiaticum Linne var. japonicum has long been used as a rheumatic remedy, as an anti-pyretic and as an anti-ulcer treatment, and for the alleviation of local pain and fever in Korea and Malaysia. In order to investigate the possibility of Crinum asiaticum Linne var. japonicum extract as a cosmetic ingredient, we measured its anti-inflammatory effect by its inhibition of iNOS (inducible nitric oxide synthase) and the release of PGE2, IL-6, and IL-8. We also measured its anti-allergic effect by its inhibition of beta-hexosamidase release. An HPLC experiment after extraction with 95% EtOH at pH 3.5 showed that Crinum asiaticum Linne var. japonicum was mainly composed of lycorine (up to 1%), a well-known immunosuppressor. The content of lycorine varied, depending on the type of plant tissue analyzed and the extraction method. In an anti-inflammatory assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)-activated mouse macrophage RAW 264.7 cells, the ethanol extract of Crinum asiaticum showed an inhibitory activity of NO production in a dose-dependent manner (IC50 = 58.5 microg/ml). Additional study by RT-PCR demonstrated that the extract of Crinum asiaticum significantly suppressed the expression of the iNOS gene. Moreover, the extract of Crinum asiaticum did not show any cytotoxicity, but did show a cell proliferation effect against LPS (a 10 approximately 60% increase in cell viability). In an assay to determine inhibition of the H2O2-activated release of PGE2, IL-6, and IL-8 in human normal fibroblast cell lines, the release of PGE2 and IL-6 was almost completely inhibited above concentrations of 0.05% and 1%, respectively. Moreover, the release of IL-8 was completely inhibited over the entire range of concentration (>0.0025%). In order to investigate the skin-sensitizing potentials of the extract of Crinum asiaticum, a human clinical test was performed after repeated epicutaneous 48-h applications under an occlusive patch (RIPT). The repeated and single cutaneous applications of Crinum asiaticum Linne var. japonicum extract under the occlusive patch did not provoke any cumulative irritation and sensitization reactions. The result showed that the extract of Crinum asiaticum Linne var. japonicum has a sufficient anti-inflammatory effect. Therefore, Crinum asiaticum Linne var. japonicum extract may be useful for development as an ingredient in cosmetic products.

The immunoreactivity and activity of adenylate cyclase type I are changed in the hippocampal CA1 region after transient forebrain ischemia in gerbils.

Adenylate cyclase (AC) has a specific sensitivity to Ca2+/calmodulin. AC-I, one of the mediator of learning and memory, plays an important role in signal transduction underlying learning and memory function. In the present study, we found ischemia-related changes of AC-I in the hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, AC-I immunoreactive neurons were detected in pyramidal and non-pyramidal cells in the hippocampus proper. AC-I immunoreactivity was significantly increased at 3 h in the CA1 region after ischemic insult. Thereafter, AC-I immunoreactivity was gradually decreased. Four days after ischemic insult, AC-I-immunoreactive CA1 pyramidal cells in the stratum pyramidale were very few due to delayed neuronal death. The results of Western blot analysis showed that changes of AC-I protein contents were similar to immunohistochemical data after ischemic insult. Gpp(NH)p-dependent AC-I activity in hippocampal CA1 region was not changed in all groups, while Ca2+/calmodulin-dependent AC-I activity in hippocampal CA1 region was significantly decreased 24 h after ischemia-reperfusion. These results suggest that the decrease of AC-I activity may be associated with impairment of neurodevelopment and neuroplasticity including learning and memory although the AC-I immunoreactivity was maintained 24 h postischemic group compared to that of the sham-operated group.

Hypothermia-induced changes of afferent sensory transmission to the VPM thalamus of rats and hamsters.

Effects of hypothermia on the afferent somatosensory transmission to the ventroposteromedial (VPM) thalamus were determined in anesthetized rats and hamsters. Hamsters showed a gradual suppression of afferent sensory transmission during cooling (to 18 degrees C) and disinhibition during subsequent warming of body temperature (Tb). However, rats exhibited steep inhibition from Tb 26 degrees C to complete absence of sensory transmission at Tb 20 degrees C and abrupt disinhibition during subsequent warming. Species difference at thalamic level was quite similar to our previous results in the primary somatosensory (SI) cortex, suggesting that changes of sensory transmission observed in the SI cortex may have already occurred at thalamic level. Differences between the cortex and the thalamus were observed only during deep hypothermia in rat and during the final period of warming in hamster. Conduction latencies of thalamocortical system of both species were not influenced during Tb lowering until 24 degrees C (equivalent to brain temperature 25-26 degrees C). These results suggest inherently different adaptability to hypothermia in processing somatosensory information between hibernator and non-hibernator, but similar sustainability of sensory functions of the thalamocortical system during hypothermia in both species.