Brown adipose tissue in obesity: Fractalkine-receptor dependent immune cell recruitment affects metabolic-related gene expression

Brown adipose tissue (BAT) plays essential role in metabolic- and thermoregulation and displays morphological and functional plasticity in response to environmental and metabolic challenges. BAT is a heterogeneous tissue containing adipocytes and various immune-related cells, however, their interaction in regulation of BAT function is not fully elucidated. Fractalkine is a chemokine synthesized by adipocytes, which recruits fractalkine receptor (CX3CR1)-expressing leukocytes into the adipose tissue. Using transgenic mice, in which the fractalkine receptor, Cx3cr1 gene was replaced by Gfp, we evaluated whether deficiency in fractalkine signaling affects BAT remodeling and function in high-fat-diet - induced obesity. Homo- and heterozygote male CX3CR1-GFP mice were fed with normal or fat enriched (FatED) diet for 10 weeks. Interscapular BAT was collected for molecular biological analysis. Heterozygous animals in which fractalkine signaling remains intact, gain more weight during FatED than CX3CR1 deficient gfp/gfp homozygotes. FatED in controls resulted in macrophage recruitment to the BAT with increased expression of proinflammatory mediators (Il1a, b, Tnfa and Ccl2). Local BAT inflammation was accompanied by increased expression of lipogenic enzymes and resulted in BAT “whitening”. By contrast, fractalkine receptor deficiency prevented accumulation of tissue macrophages, selectively attenuated the expression of Tnfa, Il1a and Ccl2, increased BAT expression of lipolytic enzymes (Atgl, Hsl and Mgtl) and upregulated genes involved thermo-metabolism (Ucp1, Pparg Pgc1a) in response to FatED. These results highlight the importance of fractalkine-CX3CR1 interaction in recruitment of macrophages into the BAT of obese mice which might contribute to local tissue inflammation, adipose tissue remodeling and regulation of metabolic-related genes.     

Interleukin 6 Deficiency Modulates the Hypothalamic Expression of Energy Balance Regulating Peptides during Pregnancy in Mice

Pregnancy is associated with hyperphagia, increased adiposity and multiple neuroendocrine adaptations. Maternal adipose tissue secretes rising amounts of interleukin 6 (IL6), which acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. To explore the role of IL6 in the central mechanisms governing dam''s energy homeostasis, early, mid and late pregnant (gestational days 7, 13 and 18) wild-type (WT) and Il6 knockout mice (Il6-KO) were compared with virgin controls at diestrus. Food intake, body weight and composition as well as indirect calorimetry measurements were performed in vivo. Anabolic and orexigenic peptides: neuropeptide Y (Npy) and agouti-related peptide (Agrp); and catabolic and anorectic neuropeptides: proopiomelanocortin (Pomc), corticotrophin and thyrotropin-releasing hormone (Crh and Trh) mRNA levels were determined by in situ hybridization. Real time-PCR and western-blot were used for additional tissue gene expression and protein studies. Non-pregnant Il6-KO mice were leaner than WT mice due to a decrease in fat but not in lean body mass. Pregnant Il6-KO mice had higher fat accretion despite similar body weight gain than WT controls. A decreased fat utilization in absence of Il6 might explain this effect, as shown by increased respiratory exchange ratio (RER) in virgin Il6-KO mice. Il6 mRNA levels were markedly enhanced in adipose tissue but reduced in hypothalamus of mid and late pregnant WT mice. Trh expression was also stimulated at gestational day 13 and lack of Il6 blunted this effect. Conversely, in late pregnant mice lessened hypothalamic Il6 receptor alpha (Il6ra), Pomc and Crh mRNA were observed. Il6 deficiency during this stage up-regulated Npy and Agrp expression, while restoring Pomc mRNA levels to virgin values. Together these results demonstrate that IL6/IL6Ra system modulates Npy/Agrp, Pomc and Trh expression during mouse pregnancy, supporting a role of IL6 in the central regulation of body fat in this physiological state.     

Intestine-specific ablation of the Hepatocyte Nuclear Factor 4a (Hnf4a) gene in mice has minimal impact on serum lipids and ileum gene expression profile due to upregulation of its paralog Hnf4g

Ablation of the gene encoding the nuclear receptor Hepatocyte Nuclear Factor 4a (Hnf4a) in the liver strongly affects HDL concentration, structure and functionality but the role of this receptor in the intestine, the second organ contributing to serum HDL levels, has been overlooked. In the present study we show that mice with intestine-specific ablation of Hnf4a (H4IntKO) had undetectable levels of ΗΝF4A in ileum, proximal and distal colon but normal expression in liver. H4IntKO mice presented normal serum lipid levels, HDL-C and particle size (α1-α3). The expression of the major HDL biogenesis genes Apoa1, Abca1, Lcat was not affected but there was significant increase in Apoc3 as well as in Hnf4g, a paralog of Hnf4a. RNA-sequencing identified metabolic pathways significantly affected by Hnf4a ablation such as type II diabetes, glycolysis, gluconeogenesis and p53 signaling. Chromatin immunoprecipitation assays showed that HNF4G bound to various apolipoprotein gene promoters in control mice but its binding affinity was reduced in the ileum of H4IntKO mice suggesting a redundancy but also a cooperation between the two factors. In the distal colon of H4IntKO mice, where both HNF4A and HNF4G are absent and in a mouse model of DSS-induced colitis presenting decreased levels of HNF4A, most lipoprotein genes were strongly downregulated. In conclusion, Hnf4a ablation in mice does not significantly affect serum lipid levels or lipoprotein gene expression in ileum possibly due to compensatory effects by its paralog Hnf4g in this tissue.     

A Gain-of-Function Mutation in Tnni2 Impeded Bone Development through Increasing Hif3a Expression in DA2B Mice

Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2^K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2^K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2^K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2^K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.     

早期与晚期应用肺表面活性物质联合经鼻持续气道正压通气治疗新生儿呼吸窘迫综合征的比较研究

目的:比较早期与晚期应用肺表面活性物质(Ps)联合经鼻持续气道正压通气(NCPAP)治疗新生儿呼吸窘迫综合征(NRDS)的临床疗效。方法:选取海南省三亚市人民医院于2014年1月～2018年7月期间收治的NRDS患儿100例,根据随机数字表法将患儿分为对照组(n=50)和研究组(n=50),对照组给予NCPAP治疗,研究组在对照组基础上联合Ps治疗,并根据Ps注入时间的不同将研究组患儿分为早期组(出生6h内注入,n=25)和晚期组(出生6～12h注入,n=25)。观察并比较对照组、早期组、晚期组患儿的临床疗效,并比较三组患儿治疗前、治疗1d后动脉血酸碱度(pH)、动脉二氧化碳分压(PCO_2)、动脉血氧分压(PO_2)、呼气末正压通气(PEEP)、吸入氧浓度(FiO_2)以及并发症发生情况。结果:早期组患儿临床总有效率高于对照组、晚期组(P0.05);而对照组、晚期组患儿临床总有效率比较差异无统计学意义(P0.05)。治疗1d后,三组患儿PCO_2较治疗前降低,PO_2较治疗前升高,且早期组PCO_2低于对照组、晚期组,PO_2高于对照组、晚期组(P0.05);但对照组、晚期组PCO_2、PO_2比较差异无统计学意义(P0.05)。治疗1d后,三组患儿PEEP、FiO_2较治疗前降低,且早期组低于对照组、晚期组(P0.05);但对照组、晚期组PEEP、Fi O2比较差异无统计学意义(P0.05)。三组患儿并发症总发生率比较差异无统计学意义(P0.05)。结论:与晚期相比,早期应用Ps联合NCPAP治疗NRDS疗效确切,其改善患儿血气指标及NCPAP参数的效果更佳,同时不会增加不良反应。     

Synthesis of Annonaceous Acetogenins from Muricatacin

Synthetic studies of annonaceous acetogenins starting from (?)-muricatacin (1a) or (+)-muricatacin are described, involving (?)-muricatacin (1a), mono-THF acetogenin, solamin (2), reticulatacin (3), (15R, 16R, 19S, 20S)-cis-solamin (4a) and (15S, 16S, 19R, 20R)-cis-solamin (4b), non-adjacent bis-THF acetogenin, 4-deoxygigantecin (5), and epoxide-bearing acetogenin, (15S, 16R, 19S, 20R)-diepomuricanin (6a).     

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi’s virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.     

Up-regulation of Arl4a gene expression by broccoli aqueous extract is associated with improved spermatogenesis in mouse testes

Introduction: Broccoli (Brassica oleracea) is well known for its properties as an anticancer, antioxidant, and scavenger of free radicals. However, its benefits in enhancing spermatogenesis have not been well established.Objective: To study broccoli aqueous extract effects on sperm factors and the expression of genes Catsper1, Catsper2, Arl4a, Sox5, and Sox9 in sperm factors in mice.Material and methods: Male mice were divided randomly into six groups: (1) Control; (2) cadmium (3 mg/kg of mouse body weight); (3) orally treated with 200 µl broccoli aqueous extract (1 g ml^-1); (4) orally treated with 400 µl of broccoli aqueous extract; (5) orally treated with 200 broccoli aqueous extract plus cadmium, and (6) orally treated with 400 µl of broccoli aqueous extract plus cadmium. We analyzed the sperms factors and Catsper1, Catsper2, Arl4a, Sox5, and Sox9 gene expression.Results: An obvious improvement in sperm count and a slight enhancement in sperm motility were observed in mice treated with broccoli extract alone or with cadmium. Sperm viability was reduced by broccoli extract except for the 200 µl dose with cadmium, which significantly increased it. Interestingly, Arl4a gene expression increased in the 400 µl broccoli- treated group. Likewise, the Arl4a mRNA level in mice treated with cadmium and 200 µl of broccoli extract was higher than in the cadmium-treated mice. Furthermore, broccoli extract enhanced the mRNA level of Catsper2 and Sox5 genes in mice treated with 200 µl and 400 µl broccoli extract plus cadmium compared with the group treated solely with cadmium.Conclusion: The higher sperm count in broccoli-treated mice opens the way for the development of pharmaceutical products for infertile men.     

Synthesis and Antimicrobial Activity of Chiral cis-Cycloheximide Isomers

Such (+)- and (?)-cis-cycloheximide isomers as isocyclohcximide (1a, 1b), α-epiisocycloheximide (2a, 2b) and neocycloheximide (3a, 3b) were synthesized by aldol condensation of (?)-(2R, 4R)- and (+)-(2S, 4S)-cis-2,4-dimethyl-1-cyclohexanone (5a, 5b). obtained by microbial resolution, with 4-(2-oxoethyl)-2,6-piperidinedione (7). The absolute configuration of the (?)-cis-ketone 5a was confirmed by chemical correlation with natural (2S, 4S, 6S, αR)-cycloheximide (4). The newly synthesized isomer, (?)-α-epiisocycloheximide (2b), showed strong antimicrobial activity against S. cerevisiae andP. oryzae close to that of natural cycloheximide (4).