Discovery of novel chiral diazepines as bombesin receptor subtype-3 (BRS-3) agonists with low brain penetration

The discovery and optimization of a novel series of BRS-3 agonists are described. We explored a potent BRS-3 agonist with low brain penetration to avoid an adverse effect derived from central nervous system exposure. Through the derivatization process, chiral diazepines 9f and 9g were identified as possessing low brain penetration as well as potent in vitro activity against human and mouse BRS-3s.     

Pyridinesulfonylureas and pyridinesulfonamides as selective bombesin receptor subtype-3 (BRS-3) agonists

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor belonging to the subfamily of bombesin-like receptors. BRS-3 is implicated in the development of obesity and diabetes. We report here small-molecule agonists that are based on a 4-(alkylamino)pyridine-3-sulfonamide core. We describe the discovery of 2a, which has mid-nanomolar potency, selectivity for human BRS-3 versus the other bombesin-like receptors, and good bioavailability.     

The design of a new potent and selective ligand for the orphan bombesin receptor subtype 3 (BRS3).

Extensive SAR studies on the unselective BRS3 agonist, [H-D-Phe6,beta-Ala11,Phe13,Nle14]-bombesin-(6-14)-nonapeptide amide, have highlighted structural features important for BRS3 activity and have provided guidance as to the design of selective agonists. A radically modified heptapeptide agonist, maintaining only the Trp-Ala moiety of the parent [H-D-Phe6,betaAla11,Phe13,Nle14]-peptide amide, and with a very different carboxyl terminal region, has been produced which was potent at BRS3 and essentially had no NMB or GRP receptor activity. Its structure is Ac-Phe-Trp-Ala-His(tauBzl)-Nip-Gly-Arg-NH2.     

心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的影响*

目的:探讨心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的作用。方法:雄性无特定病原体(SPF)级20只昆明小鼠随机分2组(每组10只),即慢性束缚应激(Stress)组和正常对照(Control)组。Stress组小鼠每天在自制式束缚器中限制活动2 h,其余时间两组小鼠在相同环境中自由饮水摄食,实验持续14 d,取血和白色脂肪组织(WAT);观察脂肪组织病理学改变,检测WAT中黄嘌呤氧化酶(XO)和烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(Nox-4)的蛋白水平,检测WAT组织中XO、Nox-4、超氧化物歧化酶(Mn SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、脂联素(ADPN)、单核细胞趋化蛋白1(MCP-1)、白介素6(IL-6)、肿瘤坏死因子α(TNF-α)、胰岛素受体底物1(IRS-1)、葡萄糖转运蛋白4(GLUT-4)、组织因子(TF)、纤溶酶原激活物抑制物1(PAI-1)的mRNA表达,检测血清和WAT组织中XO酶活性以及血清甘油三酯(TG)、总胆固醇(T-Cho)、游离脂肪酸(FFA)、尿酸(UA)的含量。结果:与control组比较,stress小鼠腹股沟WAT组织中XO免疫染色阳性着色细胞黄褐色沉淀深且丰富,WAT中出现大量的单核细胞、中性粒细胞、嗜酸性粒细胞及浆细胞浸润反应和炎症性的改变;血清XO浓度、WAT组织中XO mRNA水平和XO的酶活性显著升高(P＜0.01),血清游离脂肪酸(FFA)和尿酸(UA)的含量显著增高(P＜0.01),WAT组织中Nox-4蛋白、MCP-1、IL-6、TNF-α、TF、PAI-1mRNA的表达水平显著增高(P＜0.01),而Mn-SOD、GSH-Px、CAT、ADPN、IRS-1和GLUT-4的mRNA水平则显著降低(P＜0.01)。结论:心理应激可诱发脂肪XO过量表达及其活性增高,进而引起脂肪炎症、糖代谢及凝血酶原异常等反应。     

Cold-induced changes in brown adipose tissue thermogenic capacity of immunocompetent and immunodeficient hairless mice

Mild cold acclimation (22°C, 3 weeks) of hairless mice was shown to increase 5-fold the brown adipose tissue uncoupling protein content in immunodeficient BALB/c nu/nu mice, but by only 2.3-fold in immunocompetent BFU mice. The difference in activation of brown adipose tissue thermogenic capacity was due to a 2-fold increase in the content of brown adipose tissue in nu/nu mice only, which was paralleled by an increase in brown adipose tissue protein but not DNA content. Likewise, only in nu/nu mice the cold acclimation increased the reaction of natural killer cells in blood and peritoneal exudate with a shift from spleen to lymph nodes and increased the phagocytic index. The results indicate that the immune system may influence the defence against cold at the level of brown adipose tissue thermogenesis.Abbreviations AU arbitrary unit(s) - bw body weight - HEMA 2-hydromethyl-metacrylate copolymer - BAT brown adipose tissue - UCP uncoupling protein - ATPase mitochondrial F_oF₁-ATPsynthase - IL-1 interleukin 1 - TNF tumour necrosis factor - NK cells natural killer cells - T _a ambient temperature     

Evidence that triiodothyronine decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue

Conflicting results have been reported regarding the effect of triiodothyronine (T(3)) on serum leptin and adipose tissue leptin gene expression in human and animals. The aim of the present study was to evaluate the effect of administration of increasing doses of T(3) on serum leptin concentration and on leptin mRNA abundance in white adipose tissue of rats. The results presented in this paper indicate that administration of single different doses of T(3) to euthyroid rats resulted dose dependent increases of serum total T(3) concentrations which are associated with a decrease in white adipose tissue leptin mRNA level. The leptin mRNA level in white adipose tissue was negatively correlated with serum total T(3) concentration (r=-0.8, p<0.001). Like white adipose tissue leptin mRNA level, serum leptin concentration decreased after T(3) administration, and was also negatively correlated with the serum T(3) concentration (r=-0.8, p<0.001). In contrast, administration of T(3) to the same rats led to a significant increase in white adipose tissue expression of the malic enzyme gene (malic enzyme activity and malic enzyme mRNA level), a known target gene for T(3). The results indicate that T(3) exerts a selective inhibitory effect on white adipose tissue leptin gene expression in vivo. A conclusion is that T(3) decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue.     

猪雌激素受体关联受体a基因的克隆、表达及其对脂肪细胞脂质聚集的影响

雌激素受体关联受体α(Estrogen-related receptor α,ERRα)是调控机体能量代谢的关键转录调控因子,其在白色脂肪组织中的作用尚不清楚。本研究旨在通过touch down-PCR方法克隆猪ERRα基因的ORF序列;通过Western blotting和细胞免疫荧光染色法分析其在猪各组织及成熟脂肪细胞中的表达模式;利用ERRα特异性抑制剂XCT790处理原代培养的猪成熟脂肪细胞,探讨其对成熟脂肪细胞甘油三酯聚集的影响。结果显示,所克隆的猪ERRα基因ORF序列长1269bp(GenBank Accession No.FJ446485,尚未公布),编码422个氨基酸,其核苷酸和氨基酸序列与其他物种高度同源;ERRα蛋白高表达于猪白色脂肪组织(White adipose tissue,WAT)、肾脏以及心脏中,在脾脏中表达量较低;细胞免疫荧光化学染色显示,ERRα蛋白广泛分布于脂肪细胞的细胞核和胞浆中;XCT790在10μmol/L浓度时显著抑制了ERRα蛋白的表达和成熟脂肪细胞中甘油三酯的聚集。本研究将为有效调控体脂沉积提供新的靶点和理论参考。     

Attenuation of obesity-induced adipose tissue inflammation in C3H/HeJ mice carrying a Toll-like receptor 4 mutation

Obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. We have provided in vitro evidence that saturated fatty acids, which are released from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for Toll-like receptor 4 (TLR4) to induce the inflammatory changes in macrophages. Here we show the attenuation of adipose tissue inflammation in C3H/HeJ mice carrying a functional mutation in the TLR4 gene relative to control C3H/HeN mice during a 16-week high-fat diet. We also find that adiponectin mRNA expression is significantly reduced by co-culture of hypertrophied 3T3-L1 adipocytes and C3H/HeN peritoneal macrophages, which is reversed, when co-cultured with C3H/HeJ peritoneal macrophages. This study provides in vivo evidence that TLR4 plays a role in obesity-related adipose tissue inflammation and thus helps to identify the therapeutic targets that may reduce obesity-induced inflammation and the metabolic syndrome.     

Compensatory increase in lipogenic gene expression in adipose tissue of transgenic mice expressing constitutively active AMP-activated protein kinase-alpha1 in liver

We previously described a line of transgenic mice selectively expressing constitutively active AMPK-α1 under the control of liver-specific human apoE promoter with the hepatic control region sequence. In the short-term activation, the CA-AMPK-α1 transgenic mice at age 10–12 weeks exhibited normal hepatic triglyceride content as compared to wild-type mice due to compensatory increase in mRNA expression of genes in the cholesterol and fatty acid synthesis pathways. But it was not known whether the lipogenic gene expression in white adipose tissue also changed. Here we characterized mRNA expression profile of main lipogenic genes in the cholesterol and fatty acid biosynthesis pathway in white adipose tissue. The data show that short-term chronic activation of AMPK in liver caused marked compensatory increase in lipogenic gene expression both in liver due to induction of Srebp-2 and in white adipose tissue due to upregulation of Srebp-1c. These results support the notion that in addition to its well-recognized function for fat storage adipose tissue can play an adaptive role in fatty acid synthesis when fatty acid synthesis is severely reduced in liver, the main lipogenic organ in mammals.     

Effect of obesity and troglitazone on expression of two glutathione peroxidases: Cellular and extracellular types in serum,kidney and adipose tissue

To determine the effect of obesity on expression of cellular- (C-) and extracellular (EC-) glutathione peroxidase (GPX) in serum, kidney and adipose tissue, we measured GPX in serum, kidneys and adipose tissue of the obese Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat and its lean counterpart (LETO). We also investigated the effect of troglitazone. Five each of OLETF and LETO rats were fed diet with or without 0.2% troglitazone for 10 days. Final body weight, kidney weight, blood glucose and serum tumor necrosis factor-α (TNF-α) level were higher in OLETF rats than in LETO rats. Serum and kidney GPX activities were higher, but adipose tissue GPX activity was lower, in OLETF rats than in LETO rats. Troglitazone treatment decreased adipose tissue GPX activity and abolished overproduction of TNF-α in OLETF rats. Immunoblot analysis, for the first time, revealed that both obesity and troglitazone suppressed the protein signals for C-GPX and EC-GPX in adipose tissue. Serum protein carbonyl groups were increased in OLETF rats and troglitazone completely blocked this increase. Increased serum GPX activity in obese rat was due to the increased secretion of EC-GPX from the kidney. Troglitazone protected against the enhanced oxidative stress induced by obesity independently of the serum GPX concentration.     

Comparison of microRNAs in the intramuscular adipose tissue from Jinhua and Landrace pigs

MicroRNA (miRNA) is critically involved in lipogenesis occurring in various body parts of humans and animals. In this study, to further investigate the role and distribution of miRNA in porcine intramuscular adipose tissue, small RNAs were extracted from Jinhua and Landrace pigs to identify the expression profiles of miRNAs. miRNA expression profiles revealed that 558 miRNAs including 287 known and 271 novel miRNAs were identified, and 220 of them showed differential expression in the pigs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggested that the target genes of the differentially expressed miRNAs were involved in fatty metabolism. In conclusion, the current study reveals the active participation of miRNAs in the regulation of adipogenesis in the intramuscular adipose tissue of Jinhua and Landrace pigs.     

低氧对肥胖小鼠棕色脂肪组织相关基因表达的影响及其机制

目的：分析肥胖小鼠在低氧暴露后棕色脂肪组织的差异表达基因及通路,以探讨低氧影响棕色脂肪组织活化的机制。方法：30只雄性C57BL/6J小鼠,其中8只为普通对照组（N,n=8）;其余饲喂高脂饲料8周后,肥胖建模成功小鼠随机分为两组：肥胖对照组（OB,n=8）和肥胖低氧组（H,n=8）。H组进行11.2%氧浓度8 h/d,6天/周共4周的低氧暴露。4周后,测试血糖、血脂,取肩胛处棕色脂肪组织进行mRNA表达谱芯片扫描和生物信息学分析。利用KOBAS2.0软件对所筛选差异表达基因,并对参与关键生物过程和信号通路的差异基因进行实时荧光qPCR验证。结果：干预结束后,H组较OB组体重和血脂血糖水平显著降低;OB组较N组的上调差异基因802个,下调1 175个,差异基因的功能主要集中在糖脂合成代谢及免疫炎症反应过程;H组较OB组上调基因297个,下调228个,主要参与的生物过程有糖脂代谢、脂质转运过程、肌肉组织发育过程及脉管系统发育过程;低氧暴露调节肥胖机体棕色脂肪的通路主要集中在HIF-1、PI3K-Akt、FoxO和ErbB信号通路等过程。结论：11.2%氧气暴露可通过调节一系列棕色脂肪相关基因表达而提高棕色脂肪活性,从而下调肥胖机体体重。     

Developmental patterns of serum leptin levels, leptin gene expression in adipose tissue and Ob-Rb gene expression in hypothalamus of Erhualian and Large White pigs

~~Developmental patterns of serum leptin levels, leptin gene expression in adipose tissue and Ob-Rb gene expression in hypothalamus of Erhualian and Large White pigs~~     

Age-related changes to adipose tissue and peripheral neuropathy in genetically diverse HET3 mice differ by sex and are not mitigated by rapamycin longevity treatment

Neural communication between the brain and adipose tissues regulates energy expenditure and metabolism through modulation of adipose tissue functions. We have recently demonstrated that under pathophysiological conditions (obesity, diabetes, and aging), total subcutaneous white adipose tissue (scWAT) innervation is decreased (‘adipose neuropathy’). With advanced age in the C57BL/6J mouse, small fiber peripheral nerve endings in adipose tissue die back, resulting in reduced contact with adipose-resident blood vessels and other cells. This vascular neuropathy and parenchymal neuropathy together likely pose a physiological challenge for tissue function. In the current work, we used the genetically diverse HET3 mouse model to investigate the incidence of peripheral neuropathy and adipose tissue dysregulation across several ages in both male and female mice. We also investigated the anti-aging treatment rapamycin, an mTOR inhibitor, as a means to prevent or reduce adipose neuropathy. We found that HET3 mice displayed a reduced neuropathy phenotype compared to inbred C56BL/6 J mice, indicating genetic contributions to this aging phenotype. Compared to female HET3 mice, male HET3 mice had worse neuropathic phenotypes by 62 weeks of age. Female HET3 mice appeared to have increased protection from neuropathy until advanced age (126 weeks), after reproductive senescence. We found that rapamycin overall had little impact on neuropathy measures, and actually worsened adipose tissue inflammation and fibrosis. Despite its success as a longevity treatment in mice, higher doses and longer delivery paradigms for rapamycin may lead to a disconnect between life span and beneficial health outcomes.     

Continuous flow microcalorimetric measurement of heat production in white adipose tissue from obese (ob/ob) mice and their lean littermates

Heat production, free fatty acid and glycerol release from white adipose tissue fat pads from obese (ob/ob) mice and their lean littermates are determined. Heat production was significantly lower in obese mice compared to lean mice when expressed on wet weight basis but not when expressed on DNA basis. Noradrenaline significantly increased the heat production in fat pads from both groups of animals. However, the increase in heat production due to noradrenaline addition in fat pads from lean mice was significantly higher than in fat pads from obese mice. The release of free fatty acids and glycerol before incubation with noradrenaline was similar from fat pads from both groups of animals. Addition of noradrenaline to the fat pads increased the release of free fatty acids and glycerol in both groups of animals, but the increase was significantly larger from fat pads from lean mice. In the absence of noradrenaline the free fatty acid/glycerol ratio (mol/mol) in the effluent was 7.9:1 and 4.8:1 for lean mice and obese mice, respectively. In the presence of noradrenaline the ratio decreased to 3:1 for both groups of animals.     

皖南花猪不同发育阶段脂肪组织脂联素及受体和瘦素mRNA的变化

目的:研究皖南花猪不同发育阶段不同部位脂肪组织中脂联素(Adp)及其受体(AdpR1、AdpR2)和瘦素(leptin)mRNA的变化及性别差异。方法:选择出生、30、45、90、180日龄的皖南花猪雌、雄各5头,以β-actin为内标,采用△△Ct相对定量实时荧光PCR方法对皮下脂肪和肾周脂肪中Adp、AdpR1、AdpR2和leptin mRNA进行定量分析。结果:不同发育阶段皮下脂肪和肾周脂肪Adp、AdpR1、AdpR2、leptin mRNA的表达都有极显著差异(P<0.01)。总体上Adp mRNA在肾周脂肪显著高于皮下脂肪(P<0.05);AdpR1、AdpR2和leptin mRNA在皮下脂肪显著或极显著高于肾周脂肪(P<0.05或P<0.01)。除个别基因和个别日龄外,总体上各基因mRNA表达的性别差异不明显。无论在皮下脂肪还是肾周脂肪,Adp mRNA的表达与AdpR1、AdpR2呈显著或极显著正相关(P<0.05或P<0.01),与leptin显著负相关(P<0.05)。结论:皖南花猪不同发育阶段脂肪组织中Adp、AdpR1、AdpR2、leptin的基因表达有差异,且有组织特异性;Adp与其受体mRNA表达有相关性。