Quantitative analysis of metabolic fluxes in brown fat and skeletal muscle during thermogenesis.

Adaptive thermogenesis by brown adipose tissue (BAT) dissipates calories as heat, making it an attractive anti-obesity target. Yet how BAT contributes to circulating metabolite exchange remains unclear. Here, we quantified metabolite exchange in BAT and skeletal muscle by arteriovenous metabolomics during cold exposure in fed male mice. This identified unexpected metabolites consumed, released and shared between organs. Quantitative analysis of tissue fluxes showed that glucose and lactate provide ~85% of carbon for adaptive thermogenesis and that cold and CL316,243 trigger markedly divergent fuel utilization profiles. In cold adaptation, BAT also dramatically increases nitrogen uptake by net consuming amino acids, except glutamine. Isotope tracing and functional studies suggest glutamine catabolism concurrent with synthesis via glutamine synthetase, which avoids ammonia buildup and boosts fuel oxidation. These data underscore the ability of BAT to function as a glucose and amino acid sink and provide a quantitative and comprehensive landscape of BAT fuel utilization to guide translational studies.

A brown fat-enriched adipokine Adissp controls adipose thermogenesis and glucose homeostasis.

The signaling mechanisms underlying adipose thermogenesis have not been fully elucidated. Particularly, the involvement of adipokines that are selectively expressed in brown adipose tissue (BAT) and beige adipocytes remains to be investigated. Here we show that a previously uncharacterized adipokine (UPF0687 protein / human C20orf27 homolog) we named as Adissp (Adipose-secreted signaling protein) is a key regulator for white adipose tissue (WAT) thermogenesis and glucose homeostasis. Adissp expression is adipose-specific and highly BAT-enriched, and its secretion is stimulated by ß3-adrenergic activation. Gain-of-functional studies collectively showed that secreted Adissp promotes WAT thermogenesis, improves glucose homeostasis, and protects against obesity. Adipose-specific Adissp knockout mice are defective in WAT browning, and are susceptible to high fat diet-induced obesity and hyperglycemia. Mechanistically, Adissp binds to a putative receptor on adipocyte surface and activates protein kinase A independently of ß-adrenergic signaling. These results establish BAT-enriched Adissp as a major upstream signaling component in thermogenesis and offer a potential avenue for the treatment of obesity and diabetes.

Isolation, pathogenesis, and genetic evolution of a porcine bocavirus PBoV/HB/30/2018 strain in China.

Porcine bocavirus (PBoV) was first identified in Sweden in 2009. Due to its association with healthy as well as diseased pigs, its role in clinical disease has not been reported yet. In the present study, bocavirus was identified from the intestinal content of a 30-day-old piglet and its whole genome was constructed and phylogenetic analysis was carried on. The pathogenesis of bocavirus was investigated following orogastric inoculation of the colostrum-deprived newborn piglet with bacteria free intestinal content. The bocavirus-inoculated piglets developed diarrhea, shed virus in the rectal swabs from 18 h post inoculation and developed macroscopic and microscopic lesions in small intestine with virus confirmed by conventional PCR. This study experimentally confirmed pathogenicity and characterized bocavirus as the etiological agent of diarrhea in the colostrum-deprived newborn piglets. On phylogenetic analysis, it was observed that this virus has long evolutionary history with subsequent mutation as well as better host adaptation. This study highlights the importance of identifying bocavirus as the etiological agent of viral diarrhea that could threaten livestock, public health as well as economic loss.

Drp1-Mediated Mitochondrial Metabolic Dysfunction Inhibits the Tumor Growth of Pituitary Adenomas.

Metabolic changes have been suggested to be a hallmark of tumors and are closely associated with tumorigenesis. In a previous study, we demonstrated the role of lactate dehydrogenase in regulating abnormal glucose metabolism in pituitary adenomas (PA). As the key organelle of oxidative phosphorylation (OXPHOS), mitochondria play a vital role in the energy supply for tumor cells. However, few attempts have been made to elucidate mitochondrial metabolic homeostasis in PA. Dynamin-related protein 1 (Drp1) is a member of the dynamin superfamily of GTPases, which mediates mitochondrial fission. This study is aimed at investigating whether Drp1 affects the progression of PA through abnormal mitochondrial metabolism. We analyzed the expression of dynamin-related protein 1 (Drp1) in 20 surgical PA samples. The effects of Drp1 on PA growth were assessed in vitro and in xenograft models. We found an upregulation of Drp1 in PA samples with a low proliferation index. Knockdown or inhibition of Drp1 enhanced the proliferation of PA cell lines in vitro, while overexpression of Drp1 could reversed such effects. Mechanistically, overexpressed Drp1 damaged mitochondria by overproduction of reactive oxygen species (ROS), which induced mitochondrial OXPHOS inhibition and decline of ATP production. The energy deficiency inhibited proliferation of PA cells. In addition, overexpressed Drp1 promoted cytochrome c release from damaged mitochondria into the cytoplasm and then activated the downstream caspase apoptotic cascade reaction, which induced apoptosis of PA cells. Moreover, the decreased ATP production induced by Drp1 overexpressing activated the AMPK cellular energy stress sensor and enhanced autophagy through the AMPK-ULK1 pathway, which might play a protective role in PA growth. Furthermore, overexpression of Drp1 repressed PA growth in vivo. Our data indicates that Drp1-mediated mitochondrial metabolic dysfunction inhibits PA growth by affecting cell proliferation, apoptosis, and autophagy. Selectively targeting mitochondrial metabolic homeostasis stands out as a promising antineoplastic strategy for PA therapy.

Female mice lacking ERß display excitatory/inhibitory synaptic imbalance to drive the pathogenesis of temporal lobe epilepsy.

Epilepsy is a highly prevalent and drug-refractory neurological disorder characterized by spontaneous recurrent seizures. Estrogen is identified to be proconvulsant and lowers the seizure threshold of female epilepsy. Estrogen receptor ß (ERß) has been proposed to mediate neuroprotection in epilepsy, although the underlying mechanism remains unknown. Rationale: In this study, we investigated the role of ERß in the epileptogenesis of female temporal lobe epilepsy (TLE). Methods: Immunohistochemistry, immunofluorescence, western blots, Golgi staining, 1H MRS and whole-cell patch-clamp were used to evaluate ERß expression, pathological changes, and synaptic excitation /inhibition (E/I) balance in female TLE patients and ovariectomized (OVX) chronic epileptic mice. Electroencephalogram (EEG) recordings were recorded to evaluate the epileptic susceptibility in OVX WT and ERß-/- mice. And high-throughput RNA-sequence was performed to identify differential expression genes (DEGs) which can elucidate the potential mechanism of ERß regulating the seizure susceptibility. Results: ERß expression was decreased in the brains of female TLE patients and OVX chronic epileptic mice. ERß deletion enhanced seizure susceptibility and exacerbated the imbalance of synaptic E/I in hippocampal CA1 area of OVX epileptic mice. In line with these observations, RNA-sequence data further identified glutamine ligase (GLUL) as the target of ERß involved in regulating synaptic E/I in CA1. Furthermore, ERß agonist WAY-200070 markedly suppressed epileptic phenotypes and normalized GLUL expression in CA1 region of kainic acid (KA) induced OVX chronic epileptic model. Conclusions: Our data provide novel insight into the pathogenesis of female TLE, and indicate ERß provides a new therapeutic strategy for female TLE patients.

N6-methyladenosine regulates PEDV replication and host gene expression.

Methylation of the N6 position of adenosine (m6A) is a widespread RNA modification that is critical for various physiological and pathological processes. Although this modification was also found in the RNA of several viruses almost 40 years ago, its biological functions during viral infection have been elucidated recently. Here, we investigated the effects of viral and host RNA methylation during porcine epidemic diarrhea virus (PEDV) infection. The results demonstrated that the m6A modification was abundant in the PEDV genome and the host methyltransferases METTL3 and METTL14 and demethylase FTO were involved in the regulation of viral replication. The knockdown of the methyltransferases increased PEDV replication while silencing the demethylase decreased PEDV output. Moreover, the proteins of the YTHDF family regulated the PEDV replication by affecting the stability of m6A-modified viral RNA. In particular, PEDV infection could trigger an increasement of m6A in host RNA and decrease the expression of FTO. The m6A modification sites in mRNAs and target genes were also altered during PEDV infection. Additionally, part of the host responses to PEDV infection was controlled by m6A modification, which could be reversed by the expression of FTO. Taken together, our results identified the role of m6A modification in PEDV replication and interactions with the host.

Acute porcine epidemic diarrhea virus infection reshapes the intestinal microbiota.

The intestinal microbiota is crucial to intestinal homeostasis. Porcine epidemic diarrhea virus (PEDV) is high pathogenic to intestines, causing diarrhea, even death in piglets. To investigate the detailed relationship between PEDV infection and intestinal microbiota, the composition and distribution of intestinal microbiota from pigs were first analyzed using 16S rRNA sequencing technology. The results demonstrated that the composition and distribution of microbes in different intestinal segments were quite similar between 1-week-old and 2-week-old piglets but different from 4-week-old (weaned) piglets. Then piglets at different ages were inoculated with PEDV. The results showed that the 1-week-old piglets exhibited the most severe pathogenicity comparing to the other age groups. Further investigations indicated that Lactobacillus, Escherichia coli, and Lactococcus in the intestinal microbiota of piglets were significantly changed by PEDV infection. These results strengthen our understanding of viruses influencing intestinal microbes and remind us of the potential association between PEDV and intestinal microbes.

Interleukin (IL)-21 Promotes the Differentiation of IgA-Producing Plasma Cells in Porcine Peyer's Patches via the JAK-STAT Signaling Pathway.

Secretory IgA is critical to prevent the invasion of pathogens via mucosa. However, the key factors and the mechanisms of IgA generation in the porcine gut are not well-understood. In this study, a panel of factors, including BAFF, APRIL, CD40L, TGF-ß1, IL-6, IL-10, IL-17A, and IL-21, were employed to stimulate IgM+ B lymphocytes from porcine ileum Peyer's patches. The results showed that IL-21 significantly upregulated IgA production of B cells and facilitated cell proliferation and differentiation of antibody-secreting cells. In addition, three transcripts in porcine IgA class switch recombination (CSR), germ-line transcript α, post-switch transcript α, and circle transcript α, were first amplified by (nest-)PCR and sequenced. All these key indicators of IgA CSR were upregulated by IL-21 treatment. Furthermore, we found that IL-21 predominantly activated JAK1, STAT1, and STAT3 proteins and confirmed that the JAK-STAT signaling pathway was involved in porcine IgA CSR. Thus, IL-21 plays an important role in the proliferation and differentiation of IgA-secreting cells in porcine Peyer's patches through the JAK-STAT signaling pathway. These findings provide insights into the mucosal vaccine design by regulation of IL-21 for the prevention and control of enteric pathogens in the pig industry.

Glutamine metabolism in the proliferation of GS-expression pituitary tumor cells.

OBJECTIVE: Many cancer cells cannot survive without exogenous glutamine (Gln)，however, cancer cells expressed glutamine synthetase (GS) do not have this restriction. Previous metabolomics studies have indicated that glutamine metabolism is altered during pituitary tumorigenesis. However, the main role of Gln in pituitary adenoma (PA) pathophysiology remains unknown. The aim of this study was to evaluate the expression of GS and the main role of Gln in human PAs. METHODS: We used cell proliferation assay and flow cytometry to assess the effect of Gln depletion on three different pituitary cell lines and human primary PA cells. Then investigated the expression level of Gln synthetase (GS) in 24 human PA samples. At last, we used LC-MS/MS to identify the differences in metabolites of PA cells after the blockage of both endogenous and exogenous Gln. RESULTS: PA cell lines showed different sensitivities to Gln starvation, and the sensitivity is correlated with GS expression level. GS expressed in 21 out of the 24 human PA samples. Furthermore, a positive p53 and ki-67 index was correlated with a higher GS expression level (p<0.05). Removal of both endogenous and exogenous Gln from GS-expressing PA cells resulted in blockage of nucleotide metabolism and cell cycle arrest. CONCLUSIONS: Our data indicate that GS is needed for PA cells to proliferation during Gln deprivation, and most human PA cells express GS and might have a negative response to exogenous Gln depletion. Moreover, Gln is mainly responsible for nucleotide metabolism in the proliferation of GS-expressing pituitary tumor cells.

Angustifoline inhibits human colon cancer cell growth by inducing autophagy along with mitochondrial-mediated apoptosis, suppression of cell invasion and migration and stimulating G2/M cell cycle arrest.

PURPOSE: The prime objective of the present study was to investigate the anticancer properties of angustifoline against COLO-205 human colon cancer cells. Its effects on cell autophagy, apoptosis, cell invasion and cell migration, and cell cycle arrest were also evaluated in the current study. METHODS: WST-1 assay was used to study cytotoxic effects of the compound on the cell viability. Effects on apoptosis and cell cycle arrest were evaluated by flow cytometry. In vitro wound healing assay and matrigel assay were carried out to study the effects of angustifoline on cell migration and cell invasion respectively. To confirm autophagy, we evaluated the expression of several autophagy-associated proteins using Western blot assay along with transmission electron microscopy (TEM). RESULTS: The findings indicated that angustifoline induced dose- and time-dependent cytotoxicity in COLO-205 human colon cancer cells along with inhibiting cancer cell colony formation. Angustifoline-treated cells exhibited cell shrinkage along with distortion of the normal cell morphology. Angustifoline-treated cells were also arrested in the G2/M phase of the cell cycle, showing strong dose-dependence. The compound also led to inhibition of cell migration and cell invasion. The results showed that treatment of these cells led to generation of autophagic cell vesicles. Furthermore, it was observed that the expression of Beclin-1 and LC3-II proteins was significantly upregulated in the angustifoline-administered COLO-205 cells. CONCLUSIONS: In brief, the present study hints towards the potent anticancer potential of the natural product angustifoline against COLO-205 human colon cancer cells with in depth mechanistic studies.

Cbx4 Sumoylates Prdm16 to Regulate Adipose Tissue Thermogenesis.

Transcriptional co-activator Prdm16 controls brown fat development and white fat browning, but how this thermogenic function is modulated post-translationally is poorly understood. Here, we report that Cbx4, a Polycomb group protein, is a SUMO E3 ligase for Prdm16 and that Cbx4-mediated sumoylation of Prdm16 is required for thermogenic gene expression. Cbx4 expression is enriched in brown fat and is induced in adipose tissue by acute cold exposure. Sumoylation of Prdm16 at lysine 917 by Cbx4 blocks its ubiquitination-mediated degradation, thereby augmenting its stability and thermogenic function. Moreover, this sumoylation event primes Prdm16 to be further stabilized by methyltransferase Ehmt1. Heterozygous Cbx4-knockout mice develop metabolic phenotypes resembling those of Prdm16-knockout mice. Furthermore, fat-specific Cbx4 knockdown and overexpression produce remarkable, opposite effects on white fat remodeling. Our results identify a modifying enzyme for Prdm16, and they demonstrate a central role of Cbx4 in the control of Prdm16 stability and white fat browning.

IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk.

BACKGROUND: IRX3 was recently reported as the effector of the FTO variants. We aimed to test IRX3's roles in the browning program and to evaluate the association between the genetic variants in IRX3 and human obesity. METHODS: IRX3 expression was examined in beige adipocytes in human and mouse models, and further validated in induced beige adipocytes. The browning capacity of primary preadipocytes was assessed with IRX3 knockdown. Luciferase reporter analysis and ChIP assay were applied to investigate IRX3's effects on UCP1 transcriptional activity. Moreover, genetic analysis of IRX3 was performed in 861 young obese subjects and 916 controls. RESULTS: IRX3 expression was induced in the browning process and was positively correlated with the browning markers. IRX3 knockdown remarkably inhibited UCP1 expression in induced mouse and human beige adipocytes, and also repressed the uncoupled oxygen consumption rate. Further, IRX3 directly bound to UCP1 promoter and increased its transcriptional activity. Moreover, 17 rare heterozygous missense/frameshift IRX3 variants were identified, with a significant enrichment in obese subjects (P=0.038, OR=2.27; 95% CI, 1.02-5.05). CONCLUSIONS: IRX3 deficiency repressed the browning program of white adipocytes partially by regulating UCP1 transcriptional activity. Rare variants of IRX3 were associated with human obesity.

Transcription factor Hlx controls a systematic switch from white to brown fat through Prdm16-mediated co-activation.

Browning of subcutaneous white fat (iWAT) involves several reprograming events, but the underlying mechanisms are incompletely understood. Here we show that the transcription factor Hlx is selectively expressed in brown adipose tissue (BAT) and iWAT, and is translationally upregulated by ß3-adrenergic signaling-mediated suppression of the translational inhibitor 4E-BP1. Hlx interacts with and is co-activated by Prdm16 to control BAT-selective gene expression and mitochondrial biogenesis. Hlx heterozygous knockout mice have defects in brown-like adipocyte formation in iWAT, and develop glucose intolerance and high fat-induced hepatic steatosis. Conversely, transgenic expression of Hlx at a physiological level drives a full program of thermogenesis and converts iWAT to brown-like fat, which improves glucose homeostasis and prevents obesity and hepatic steatosis. The adipose remodeling phenotypes are recapitulated by fat-specific injection of Hlx knockdown and overexpression viruses, respectively. Our studies establish Hlx as a powerful regulator for systematic white adipose tissue browning and offer molecular insights into the underlying transcriptional mechanism.The transcriptional co-activator Prdm16 regulates browning of white adipose tissue (WAT). Here, the authors show that Prdm16 interacts with the transcription factor Hlx, which is stabilized in response to ß3-adrenergic signaling, to increase thermogenic gene expression and mitochondrial biogenesis in subcutaneous WAT.

Arogenate Dehydratase Isoforms Differentially Regulate Anthocyanin Biosynthesis in Arabidopsis thaliana.

Anthocyanins, a group of L-phenylalanine (Phe)-derived flavonoids, have been demonstrated to play important roles in plant stress resistance and interactions between plants and insects. Although the anthocyanin biosynthetic pathway and its regulatory mechanisms have been extensively studied, it remains unclear whether the level of Phe supply affects anthocyanin biosynthesis. Here, we investigated the roles of arogenate dehydratases (ADTs), the key enzymes that catalyze the conversion of arogenate into Phe, in sucrose-induced anthocyanin biosynthesis in Arabidopsis. Genetic analysis showed that all six ADT isoforms function redundantly in anthocyanin biosynthesis but have differential contributions. ADT2 contributes the most to anthocyanin accumulation, followed by ADT1 and ADT3, and ADT4-ADT6. We found that anthocyanin content is positively correlated with the levels of Phe and sucrose-induced ADT transcripts in seedlings. Consistently, addition of Phe to the medium could dramatically increase anthocyanin content in the wild-type plants and rescue the phenotype of the adt1 adt3 double mutant regarding the anthocyanin accumulation. Moreover, transgenic plants overexpressing ADT4, which appears to be less sensitive to Phe than overexpression of ADT2, hyperaccumulate Phe and produce elevated level of anthocyanins. Taken together, our results suggest that the level of Phe is an important regulatory factor for sustaining anthocyanin biosynthesis.

Arabidopsis thylakoid formation 1 is a critical regulator for dynamics of PSII-LHCII complexes in leaf senescence and excess light.

In higher plants, photosystem II (PSII) is a large pigment-protein supramolecular complex composed of the PSII core complex and the plant-specific peripheral light-harvesting complexes (LHCII). PSII-LHCII complexes are highly dynamic in their quantity and macro-organization to various environmental conditions. In this study, we reported a critical factor, the Arabidopsis Thylakoid Formation 1 (THF1) protein, which controls PSII-LHCII dynamics during dark-induced senescence and light acclimation. Loss-of-function mutations in THF1 lead to a stay-green phenotype in pathogen-infected and senescent leaves. Both LHCII and PSII core subunits are retained in dark-induced senescent leaves of thf1, indicative of the presence of PSII-LHCII complexes. Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis showed that, in dark- and high-light-treated thf1 leaves, a type of PSII-LHCII megacomplex is selectively retained while the stability of PSII-LHCII supercomplexes significantly decreased, suggesting a dual role of THF1 in dynamics of PSII-LHCII complexes. We showed further that THF1 interacts with Lhcb proteins in a pH-dependent manner and that the stay-green phenotype of thf1 relies on the presence of LHCII complexes. Taken together, the data suggest that THF1 is required for dynamics of PSII-LHCII supramolecular organization in higher plants.

[Study on parentage testing in JIRONG rabbit by microsatellite markers].

Thirteen microsatellite markers, including Sat2,Sat3,Sat4,Sat5,Sat7,Sat8,Sat12, Sat13, Sat16,Sol08,Sol28,Sol30 and Sol03, were studied for their parentage-testing application in a group of 30 JIRONG Rabbits in the present report. The 13 microsatellite loci were successfully amplified with specific primers designed according to known sequences. The PCR products amplified from the microsatellite loci were analyzed by 8% denaturing polyacrylamide gel electrophoresis. The results demonstrated that the average alleles and the mean heterozygosity (Hs) and the polymorphism information content (PIC) and the combined exclusion probability (PE2) of the 13 microsatellite loci were 3.46, 0.578, 0.531, and 0.999329, respectively. The exclusion probability (PE1) of the 13 loci was 0.935226, and the confidence of the parentage testing was less than 80% when data of both parents were unknown, while the exclusion probability (PE1) and the confidence were 0.999329 and 95% respectively with known data of a single parent. Since the data of the rabbit maternal lines studied were known, the paternal lines of the group were successfully identified using the 13 microsatellite loci with high confidence.