Down-regulation of microsomal prostaglandin E2 synthase-1 in adipose tissue by high-fat feeding

Objective: Prostaglandin (PG)E₂ is a lipid mediator implicated in inflammatory diseases and in the regulation of lipolysis and adipocyte differentiation. This work was, thus, undertaken to study the regulation of the various PGE₂ synthases (PGESs) in obesity. Research Methods and Procedures: C57Bl/6 mice were subjected to a high‐fat or regular diet for 12 weeks. The levels of PGE₂ in white adipose tissue (WAT) of lean and obese mice were quantified by liquid chromatography‐mass spectrometry, and the change in expression of the three major PGES caused by diet‐induced obesity was characterized by Western blotting. Human preadipocytes and 3T3‐L1 cells were used to assess the expression of microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) during adipogenesis. Results: mPGES‐1, mPGES‐2, and cytosolic PGES proteins were all detected in WAT of lean animals. mPGES‐1 was expressed at higher levels in WAT than in any other tissues examined and was more abundant (3‐ to 4‐fold) in epididymal (visceral) compared with inguinal (subcutaneous) WAT. Expression of mPGES‐1 was also detected in undifferentiated and differentiated 3T3‐L1 cells and in human primary subcutaneous preadipocytes at all stages of adipogenesis. The mPGES‐1 protein was substantially down‐regulated in epididymal and inguinal WAT of obese mice, whereas mPGES‐2 and cytosolic PGES remained relatively stable. Concordantly, the PGE₂ levels in obese inguinal WAT were significantly lower than those of lean animals. Discussion: These data suggest that mPGES‐1 is the major form of PGESs contributing to the synthesis of PGE₂ in WAT and that its down‐regulation might be involved in the alterations of lipolysis and adipogenesis associated with obesity.     

Differential patterns of serum concentration and adipose tissue expression of chemerin in obesity: Adipose depot specificity and gender dimorphism

Chemerin, a recognized chemoattractant, is expressed in adipose tissue and plays a role in adipocytes differentiation and metabolism. Gender- and adipose tissue-specific differences in human chemerin expression have not been well characterized. Therefore, these differences were assessed in the present study. The body mass index (BMI) and the circulating levels of chemerin and other inflammatory, adiposity and insulin resistance markers were assessed in female and male adults of varying degree of obesity. Chemerin mRNA expression was also measured in paired subcutaneous and visceral adipose tissue samples obtained from a subset of the study subjects. Serum chemerin concentrations correlated positively with BMI and serum leptin levels and negatively with high density lipoprotein (HDL)-cholesterol levels. No correlation was found between serum chemerin concentrations and fasting glucose, total cholesterol, low density lipoprotein (LDL)-cholesterol, triglycerides, insulin, C-reactive protein or adiponectin. Similarly, no relation was observed with the homeostasis model assessment for insulin resistance (HOMA-IR) values. Gender- and adipose tissue-specific differences were observed in chemerin mRNA expression levels, with expression significantly higher in women than men and in subcutaneous than visceral adipose tissue. Interestingly, we found a significant negative correlation between circulating chemerin levels and chemerin mRNA expression in subcutaneous fat. Among the subjects studied, circulating chemerin levels were associated with obesity markers but not with markers of insulin resistance. At the tissue level, fat depot-specific differential regulation of chemerin mRNA expression might contribute to the distinctive roles of subcutaneous vs. visceral adipose tissue in human obesity.     

Depot-specific prostaglandin synthesis in human adipose tissue: a novel possible mechanism of adipogenesis

Despite the magnitude of the obesity epidemic, the mechanisms that contribute to increases in fat mass and to differences in fat depots are still poorly understood. Prostanoids have been proposed as potent adipogenic hormones, e.g. metabolites of prostaglandin J2 (PGJ2) bind and activate PPARγ. We hypothesize that an altered expression of enzymes in PGJ2 synthesis may represent a novel pathogenic mechanism in human obesity. We characterized adipose depot-specific expression of enzymes in PGJ2 synthesis, prostaglandin transporter and PPARγ isoforms. Paired omental and subcutaneous adipose tissue samples were obtained from 26 women undergoing elective abdominal surgery and gene expression examined in whole tissue and cultured preadipocytes using an Affymetrix cDNA microarray technique and validated with quantitative real-time PCR. All enzymes involved in prostaglandin synthesis were expressed in both adipose tissues. Expression of prostaglandin synthase-1 (PGHS1), prostaglandin D synthase (PTGDS), human prostaglandin transporter (hPGT) and PPARγ2 was higher in OM adipose tissue compared to SC, whereas 17β-hydroxysteroid dehydrogenase 5 (AKR1C3) showed predominance in SC adipose tissue. In SC adipose tissue, PGHS1 mRNA expression increased with BMI. The differential, depot-specific expression of key enzymes involved in transport, synthesis and metabolism of prostaglandins may have an important impact upon fat cell biology and may help to explain some of the observed depot-specific differences. In addition, the positive correlation between PGHS1 and BMI offers the novel hypothesis that the regulation of PG synthesis may have a role in determining fat distribution in human obesity.     

一种快速鉴定猪肺炎支原体免疫显性蛋白抗原的ELISA方法的建立

旨在建立一种快速鉴定猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)血清体液免疫显性蛋白抗原的方法。通过构建p GEX-6P-1-mhp366重组表达质粒并转入大肠杆菌BL21(DE3),将GST-Mhp366重组蛋白进行原核表达。将GST-Mhp366重组菌和GST工程菌裂解液加入谷胱甘肽包被板进行抗原包被,分别与17份Mhp阳性血清和13份Mhp阴性血清反应,通过对抗原包被量、封闭液、血清和二抗稀释度的优化,确定间接ELISA的反应条件。最终确定GST-Mhp366重组菌和GST工程菌裂解液原液为抗原的最佳包被量,PBS+10%FBS+2.5%脱脂奶粉为最佳封闭液,分别将血清按照1∶500稀释、酶标二抗按照1∶40 000稀释作为最佳工作浓度,从而建立了一种基于间接ELISA的快速鉴定Mhp血清体液免疫显性蛋白抗原的方法,同时通过已知的血清体液免疫显性蛋白Mhp156和Mhp364对所建立的方法进行了验证。该方法的建立能够用于在基因组水平高通量筛选Mhp血清体液免疫显性蛋白抗原,并为Mhp初乳体液免疫和黏膜免疫显性蛋白抗原鉴定方法的建立奠定基础。     

Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay

Objective: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme‐linked immunosorbent assay (ELISA) for direct measurement of mouse resistin. Research Methods and Procedures: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity‐purified anti‐C‐terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin‐linked horseradish peroxidase. Results: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2‐ to 5‐fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or β‐estradiol, in cultured adipocytes reduces resistin protein levels in a dose‐dependent manner. Discussion: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.     

Serum phospholipid fatty acids, adipose tissue, and metabolic markers in obese adolescents

Objective: Fatty acid (FA) composition has a role in adipogenesis. The objective was to study serum phospholipid (PL) FAs in adolescents and their relation to abdominal adipose tissue (AT) compartments and metabolic markers. Research Methods and Procedures: Abdominal AT was measured by magnetic resonance imaging and FA pattern was determined in serum PL of 10 obese adolescents (5 females), median age 12.0 years (range, 10.4 to 16.4) and BMI 30.7 (26.8 to 40.4), and 15 lean control subjects (9 females), median age 12.6 years (range, 11.3 to 15.4), and BMI 19.5 (17.1 to 23.4). Results: Obese adolescents had relatively higher levels of saturated FA (SFA) and nervonic acid compared with controls. Serum PL concentration of n‐3 polyunsaturated fatty acids (PUFA) was lower in the obese vs. lean females (p = 0.01), including docosahexaenoic acid (DHA) (p = 0.01). The ratios of arachidonic acid to DHA and total n‐6/n‐3 FA were increased in obese children (p = 0.02 and 0.01, respectively). n‐3 PUFAs were inversely correlated to all subcutaneous AT compartments except visceral AT. The homeostasis model assessment index of β‐cell function related inversely to DHA concentration (p = 0.03). All changes were more marked in the females. Discussion: Serum FA pattern in obese adolescents differed significantly from that in age‐matched lean controls, reflecting a decrease in n‐3 PUFA, especially DHA, and an increase in SFA. The subcutaneous AT, but not visceral AT, correlated to the changes in PUFA and SFA, suggesting an abnormal essential FA metabolism in obese adolescents.     

Purification, immunochemical quantification and localization in rat heart of putative fatty acid translocase (FAT/CD36)

Evidence is accumulating that the heavily glycosylated integral membrane protein fatty acid translocase (FAT/CD36) is involved in the transport of long-chain fatty acids across the sarcolemma of heart muscle cells. The aim of this study was to analyse the distribution between FAT/CD36 present in cardiac myocytes and endothelial cells. We therefore developed a method to purify FAT/CD36 from total rat heart and isolated cardiomyocytes, and used the proteins as standards in an immunochemical assay. Two steps, chromatography on wheat germ agglutinin-agarose and anion-exchange chromatography on Q-Sepharose fast flow, were sufficient for obtaining the protein in a > 95% pure form. When used to isolate FAT/CD36 from total heart tissue, the FAT/CD36 yield of the method was 9% and the purification factor was 64. Purifying FAT/CD36 from isolated cardiomyocytes yielded the same 88 kDa protein band on SDS-PAGE gels and reactivity of this band on western blots was comparable to that of the FAT/CD36 isolated from total hearts. Quantifying FAT/CD36 contents by western blotting showed that the amounts of FAT/CD36 that are present in isolated cardiomyocytes (10 ± 3 μg/mg protein) and total hearts (14 ± 4 μg/mg protein) are of comparable magnitude. Immunofluorescence labelling showed that at least a part of the FAT/CD36 present in the cardiomyocyte is associated with the sarcolemma. This study established that FAT/CD36 is a relatively abundant protein in the cardiomyocyte. In addition, the further developed purification procedure is the first method for isolating FAT/CD36 from rat heart and cardiomyocyte FAT/CD36.     

Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood

Aims: The study evaluated the efficiency of culture, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assays for the detection of Salmonella in naturally contaminated seafood. Methods and Results: In this study, 215 seafood samples comprising fish, shrimp, crab, clam, mussel, oyster, squid, cuttlefish and octopus from fish market of Cochin (India), were compared by culture, ELISA and PCR methods. Bacteriological Analytical Manual (BAM), U.S. Food and Drug Administration (USFDA) method was followed for culture assay, and Salmonella Tek, a commercial sandwich ELISA kit, was used for ELISA assay. Salmonella‐specific PCR assay was developed for 284 bp Salmonella‐specific invA gene amplicon. PCR assay exhibited 31·6% seafood positive for Salmonella followed by ELISA (23·7%) and culture method (21·3%). There was fair to excellent agreement between culture, ELISA and PCR assays (kappa coefficient values ranging from 0·385 to 1·0) for different seafood samples. Conclusion: The investigation revealed the greater concordance between culture and ELISA methods for seafood. Among the three methods, PCR assay was most sensitive. Lower detection rate with culture and ELISA assays could be attributed to greater sensitivity of the PCR method in the detection of Salmonella in seafood. Significance and Impact of the Study: We propose the incorporation of dual tests based on different principle and procedure for the routine analysis of Salmonella in seafood.     

Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake

This study investigates the role of the cytoplasmic C terminus of fatty acid translocase (FAT/CD36) in localization of the molecule to the plasma membrane, its insertion into lipid rafts, and its ability to enhance long-chain fatty acid uptake in transfected H4IIE rat hepatoma cells. In these cells, wild-type FAT/CD36 is localized to both lipid raft and nonraft domains of the plasma membrane. Interestingly, a FAT/CD36 truncation mutant lacking the final 10 amino acids of the cytoplasmic C terminus was retained within the cell in detergent-resistant membranes, and unlike wild-type FAT/CD36, it did not enhance oleate uptake. Furthermore, expression of FAT/CD36 in these cells increased the incorporation of oleate into diacylglycerol, a property that was not shared by truncated FAT/CD36. To examine whether the C terminus itself has an intrinsic ability to dictate the plasma membrane localization of FAT/CD36, this region was fused in-frame to enhanced green fluorescent protein (EGFP). This domain was sufficient to attach EGFP to cellular membranes, suggesting an involvement in the intracellular traffic of the molecule. We conclude that the C terminus of FAT/CD36 is required for localization of the receptor to the cell surface and its ability to enhance cellular oleate uptake.     

A novel procedure for protein extraction from formalin-fixed paraffin-embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Composition of adipose tissue and marrow fat in humans by 1H NMR at 7 Tesla

Proton NMR spectroscopy at 7 Tesla (7T) was evaluated as a new method to quantify human fat composition noninvasively. In validation experiments, the composition of a known mixture of triolein, tristearin, and trilinolein agreed well with measurements by (1)H NMR spectroscopy. Triglycerides in calf subcutaneous tissue and tibial bone marrow were examined in 20 healthy subjects by (1)H spectroscopy. Ten well-resolved proton resonances from triglycerides were detected using stimulated echo acquisition mode sequence and small voxel ( approximately 0.1 ml), and T(1) and T(2) were measured. Triglyceride composition was not different between calf subcutaneous adipose tissue and tibial marrow for a given subject, and its variation among subjects, as a result of diet and genetic differences, fell in a narrow range. After correction for differential relaxation effects, the marrow fat composition was 29.1 +/- 3.5% saturated, 46.4 +/- 4.8% monounsaturated, and 24.5 +/- 3.1% diunsaturated, compared with adipose fat composition, 27.1 +/- 4.2% saturated, 49.6 +/- 5.7% monounsaturated, and 23.4 +/- 3.9% diunsaturated. Proton spectroscopy at 7T offers a simple, fast, noninvasive, and painless method for obtaining detailed information about lipid composition in humans, and the sensitivity and resolution of the method may facilitate longitudinal monitoring of changes in lipid composition in response to diet, exercise, and disease.     

Preparation and Application of Monoclonal Antibodies Specific for Salicylic Acid (in English)

专一识别水杨酸的单克隆抗体的制备及应用 王树才^1,2李国婧¹ 夏凯¹ 徐朗莱²陈溥言³ 周燮^1*     

Diets high in monounsaturated and polyunsaturated fatty acids decrease fatty acid synthase protein levels in adipose tissue but do not alter other markers of adipose function and inflammation in diet-induced obese rats

This study investigates the effects of monounsaturated and polyunsaturated fatty acids from different fat sources (High Oleic Canola, Canola, Canola–Flaxseed (3:1 blend), Safflower, or Soybean Oil, or a Lard-based diet) on adipose tissue function and markers of inflammation in Obese Prone rats fed high-fat (55% energy) diets for 12 weeks. Adipose tissue fatty acid composition reflected the dietary fatty acid profiles. Protein levels of fatty acid synthase, but not mRNA levels, were lower in adipose tissue of all groups compared to the Lard group. Adiponectin and fatty acid receptors GPR41 and GPR43 protein levels were also altered, but other metabolic and inflammatory mediators in adipose tissue and serum were unchanged among groups. Overall, rats fed vegetable oil- or lard-based high-fat diets appear to be largely resistant to major phenotypic changes when the dietary fat composition is altered, providing little support for the importance of specific fatty acid profiles in the context of a high-fat diet.     

Enzyme-linked immunosorbent assay for detection of Trichinella spiralis antibodies and the surveillance of selected pig breeding farms in the Republic of Korea

Trichinellosis is a parasitic zoonosis of public health importance. It is caused by Trichinella spiralis which has a wide host range including humans. In the present communication, the ELISA technique was employed on a total of 803 blood samples from 7 selected pig breeding farms in 1996 for diagnosis and surveillance of trichinellosis. Out of the entire 803 samples, nine were found to be suspected while one was positive by ELISA. But western blot analyses employed for further confirmation have shown that all of 10 samples did not react to larval excretory-secretory product antigens. These results indicate that pig breeding farms included in the present study are free from trichinellosis. However, it does not mean Korea is free from trichinellosis since human trichinellosis has recently been reported. The necessity of continued surveillance for trichinellosis in both pigs and wild animals was discussed.     

肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA法建立及其在多糖浓度测定中的应用

目的：利用肺炎球菌1型全菌体制备多克隆抗体,并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法（ Enzyme-linked immunosorbent assay ,ELISA）,用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周,获得高滴度的抗多糖血清,经过亲和层析纯化,获得高纯度的兔抗肺炎1型多糖抗体IgG。以纯化IgG作为包被抗体,加入多糖样品,再以生物素化的抗体作为检测抗体,建立夹心ELISA法检测肺炎1型多糖浓度。确定标准曲线的最佳线性范围,并对该方法进行特异性、准确性和精密度验证。结果兔免疫血清经过双向免疫扩散检测抗体滴度可达1∶32;该方法的线性检测范围为1．56～50 ng/mL;最低检测限为3．13 ng/mL。在标准品中混入其他型别多糖或培养基,回收率分别为102％和108％;该方法批内精密度和批间精密度分别为6．08％和7．01％。结论建立的夹心ELISA方法,其特异性、准确性和精密度均良好,可以特异地检测肺炎球菌1型多糖浓度。     

Effect of plasma triglyceride metabolism on lipid storage in adipose tissue: Studies using genetically engineered mouse models

The obesity epidemic is associated with an increased incidence of type 2 diabetes, cardiovascular morbidity and various types of cancer. A better insight into the molecular mechanisms that underlie adipogenesis and obesity may result in novel therapeutic handles to fight obesity and these associated diseases. Adipogenesis is determined by the balance between uptake of fatty acids (FA) from plasma into adipocytes, intracellular FA oxidation versus esterification of FA into triglycerides (TG), lipolysis of TG by intracellular lipases, and secretion of FA from adipocytes. Here, we review the mechanisms that are specifically involved in the entry of FA into adipose tissue. In plasma, these originating FA are either present as TG within apoB-containing lipoproteins (i.e. chylomicrons and VLDL) or as free FA bound to albumin. Kinetic studies, however, have revealed that TG are the major source of FA entering adipose tissue, both in the fed and fasted condition. In fact, studies with genetically engineered mice have revealed that the activity of lipoprotein lipase (LPL) is a major determinant for the development of obesity. As a general rule, high fat diet-induced adipogenesis is aggravated by stimulated LPL activity (e.g. by adipose tissue-specific overexpression of LPL or deficiency for apoCIII), and attenuated by inhibited LPL activity (e.g. by adipose-specific deficiency for LPL, overexpression of apoCI or angptl4, or by deficiency for apoE or the VLDL receptor). In addition, we describe that the trans-membrane transport of FA and cytoplasmic binding of FA in adipocytes can also dramatically affect adipogenesis. The relevance of these findings for human pathophysiology is discussed.