Age- and islet autoimmunity-associated differences in amino acid and lipid metabolites in children at risk for type 1 diabetes.

OBJECTIVE: Islet autoimmunity precedes type 1 diabetes and often initiates in childhood. Phenotypic variation in islet autoimmunity relative to the age of its development suggests heterogeneous mechanisms of autoimmune activation. To support this notion, we examined whether serum metabolite profiles differ between children with respect to islet autoantibody status and the age of islet autoantibody development. RESEARCH DESIGN AND METHODS: The study analyzed 29 metabolites of amino acid metabolism and 511 lipids assigned to 12 lipid clusters in children, with a type 1 diabetic parent, who first developed autoantibodies at age 2 years or younger (n = 13), at age 8 years or older (n = 22), or remained autoantibody-negative, and were matched for age, date of birth, and HLA genotypes (n = 35). Ultraperformance liquid chromatography and mass spectroscopy were used to measure metabolites and lipids quantitatively in the first autoantibody-positive and matched autoantibody-negative serum samples and in a second sample after 1 year of follow-up. RESULTS: Differences in the metabolite profiles were observed relative to age and islet autoantibody status. Independent of age-related differences, autoantibody-positive children had higher levels of odd-chain triglycerides and polyunsaturated fatty acid-containing phospholipids than autoantibody-negative children and independent of age at first autoantibody appearance (P < 0.0001). Consistent with our hypothesis, children who developed autoantibodies by age 2 years had twofold lower concentration of methionine compared with those who developed autoantibodies in late childhood or remained autoantibody-negative (P < 0.0001). CONCLUSIONS: Distinct metabolic profiles are associated with age and islet autoimmunity. Pathways that use methionine are potentially relevant for developing islet autoantibodies in early infancy.

Splanchnic balance of free fatty acids, endocannabinoids, and lipids in subjects with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Animal studies suggest that endocannabinoids could contribute to the development of nonalcoholic fatty liver disease (NAFLD). In addition, NAFLD has been shown to be associated with multiple changes in lipid concentrations in liver biopsies. There are no data on splanchnic free fatty acid (FFA), glycerol, ketone body, endocannabinoid, and lipid fluxes in vivo in subjects with NAFLD. METHODS: We performed hepatic venous catheterization studies in combination with [(2)H(2)]palmitate infusion in the fasting state and during a low-dose insulin infusion in 9 subjects with various degrees of hepatic steatosis as determined using liver biopsy. Splanchnic balance of endocannabinoids and individual lipids was determined using ultra performance liquid chromatography coupled to mass spectrometry. RESULTS: Concentrations of the endocannabinoid 2-arachidonoylglycerol were higher in arterialized (91 ± 33 µg/L basally) than in hepatic venous (51 ± 19 µg/L; P < .05) plasma. Fasting arterial (r = 0.72; P = .031) and hepatic venous (r = 0.70; P = .037) concentrations of 2-arachidonoylglycerol were related positively to liver fat content. Analysis of fluxes of 85 different triglycerides showed that the fatty liver overproduces saturated triglycerides. In the plasma FFA fraction in the basal state, the relative amounts of palmitoleate and linoleate were lower and those of stearate and oleate were higher in the hepatic vein than in the artery. Absolute concentrations of all nontriglyceride lipids were comparable in arterialized venous plasma and the hepatic vein both in the basal and insulin-stimulated states. CONCLUSIONS: The human fatty liver takes up 2-arachidonoylglycerol and overproduces triacylglycerols containing saturated fatty acids, which might reflect increased de novo lipogenesis.

Dysregulation of lipid and amino acid metabolism precedes islet autoimmunity in children who later progress to type 1 diabetes.

The risk determinants of type 1 diabetes, initiators of autoimmune response, mechanisms regulating progress toward beta cell failure, and factors determining time of presentation of clinical diabetes are poorly understood. We investigated changes in the serum metabolome prospectively in children who later progressed to type 1 diabetes. Serum metabolite profiles were compared between sample series drawn from 56 children who progressed to type 1 diabetes and 73 controls who remained nondiabetic and permanently autoantibody negative. Individuals who developed diabetes had reduced serum levels of succinic acid and phosphatidylcholine (PC) at birth, reduced levels of triglycerides and antioxidant ether phospholipids throughout the follow up, and increased levels of proinflammatory lysoPCs several months before seroconversion to autoantibody positivity. The lipid changes were not attributable to HLA-associated genetic risk. The appearance of insulin and glutamic acid decarboxylase autoantibodies was preceded by diminished ketoleucine and elevated glutamic acid. The metabolic profile was partially normalized after the seroconversion. Autoimmunity may thus be a relatively late response to the early metabolic disturbances. Recognition of these preautoimmune alterations may aid in studies of disease pathogenesis and may open a time window for novel type 1 diabetes prevention strategies.

Application of lipidomics and metabolomics to the study of adipose tissue.

Role of specific reactive lipids as well as amino acids in control of insulin signalling in adipose tissue is well recognized. Since it is practically impossible to measure the levels of all metabolites in the biological sample simultaneously with a single analytical platform, we utilize multiple platforms to study the lipids and metabolites of relevance to adipose tissue metabolism and insulin signalling. Two screening platforms cover a broad range of lipid molecular species (UPLC/MS based lipidomics platform) as well as organic acids and sterols (GCxGC-TOF platform). A targeted platform for amino acids (UPLC) is also applied.

Global transcript profiles of fat in monozygotic twins discordant for BMI: pathways behind acquired obesity.

BACKGROUND: The acquired component of complex traits is difficult to dissect in humans. Obesity represents such a trait, in which the metabolic and molecular consequences emerge from complex interactions of genes and environment. With the substantial morbidity associated with obesity, a deeper understanding of the concurrent metabolic changes is of considerable importance. The goal of this study was to investigate this important acquired component and expose obesity-induced changes in biological pathways in an identical genetic background. METHODS AND FINDINGS: We used a special study design of "clonal controls," rare monozygotic twins discordant for obesity identified through a national registry of 2,453 young, healthy twin pairs. A total of 14 pairs were studied (eight male, six female; white), with a mean +/- standard deviation (SD) age 25.8 +/- 1.4 y and a body mass index (BMI) difference 5.2 +/- 1.8 kg/m(2). Sequence analyses of mitochondrial DNA (mtDNA) in subcutaneous fat and peripheral leukocytes revealed no aberrant heteroplasmy between the co-twins. However, mtDNA copy number was reduced by 47% in the obese co-twin's fat. In addition, novel pathway analyses of the adipose tissue transcription profiles exposed significant down-regulation of mitochondrial branched-chain amino acid (BCAA) catabolism (p < 0.0001). In line with this finding, serum levels of insulin secretion-enhancing BCAAs were increased in obese male co-twins (9% increase, p = 0.025). Lending clinical relevance to the findings, in both sexes the observed aberrations in mitochondrial amino acid metabolism pathways in fat correlated closely with liver fat accumulation, insulin resistance, and hyperinsulinemia, early aberrations of acquired obesity in these healthy young adults. CONCLUSIONS: Our findings emphasize a substantial role of mitochondrial energy- and amino acid metabolism in obesity and development of insulin resistance.

Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae.

BACKGROUND: Secretion stress is caused by compromised folding, modification or transport of proteins in the secretory pathway. In fungi, induction of genes in response to secretion stress is mediated mainly by the unfolded protein response (UPR) pathway. This study aims at uncovering transcriptional responses occurring in the filamentous fungi Trichoderma reesei exposed to secretion stress and comparing these to those found in the yeast Saccharomyces cerevisiae. RESULTS: Chemostat cultures of T. reesei expressing human tissue plasminogen activator (tPA) and batch bioreactor cultures treated with dithiothreitol (DTT) to prevent correct protein folding were analysed with cDNA subtraction and cDNA-amplified fragment length polymorphism (AFLP) experiments. ESTs corresponding to 457 unique genes putatively induced under secretion stress were isolated and the expression pattern of 60 genes was confirmed by Northern analysis. Expression of these genes was also studied in a strain over-expressing inositol-requiring enzyme 1 (IREI) protein, a sensor for the UPR pathway. To compare the data with that of S. cerevisiae, published transcriptome profiling data on various stress responses in S. cerevisiae was reanalysed. The genes up-regulated in response to secretion stress included a large number of secretion related genes in both organisms. In addition, analysis of T. reesei revealed up regulation of the cpc1 transcription factor gene and nucleosomal genes. The induction of the cpcA and histone gene H4 were shown to be induced also in cultures of Aspergillus nidulans treated with DTT. CONCLUSION: Analysis of the genes induced under secretion stress has revealed novel features in the stress response in T. reesei and in filamentous fungi. We have demonstrated that in addition to the previously rather well characterised induction of genes for many ER proteins or secretion related proteins also other types of responses exist.

Development of a high-throughput format for solid-phase extraction of enantiomers using an immunosorbent in 384-well plates.

An antibody-based solid-phase extraction method for filtered 384-well plates was developed for a medical drug candidate having two enantiomeric forms in order to demonstrate the potential of the use of recombinant antibody fragments as specific and efficient immunosorbents. An immobilization method using a six-histidine tag of the antibody fragment and mild oxidation was applied in order to immobilize antibody fragments in an oriented and kinetically stable way that ensured high capacity of the antibody support. Phosphate buffer or plasma spiked with enantiomers were used as samples. Selective solid-phase extraction was followed by liquid chromatography-mass spectrometry analysis. Average recoveries for buffer and plasma samples ranged from 79 to 122% and 80 to 108%, respectively. Good linearity was observed in the concentration range of 30-3000 ng/mL of the enantiomer.

Polymerization of guaiacol and a phenolic beta-O-4-substructure by Trametes hirsuta laccase in the presence of ABTS.

The reaction of the monomeric lignin model compound guaiacol and the beta-O-4-type dimer erol (1-(4-hydroxy-3-methoxyphenyl)-2(2-methoxyphenoxy)-propane-1,3-diol with laccase from Trametes hirsuta was studied in the presence of the mediator ABTS (2,2'-azino-di[3ethylbenzothiazoline-6-sulfonic acid]). The product mixtures were analyzed by means of aqueous-phase size exclusion chromatography (SEC) with 50 mM NaOH as eluent. Interestingly, in the laccase-catalyzed reaction with both substrates, the mediator not only functioned as an electron carrier but underwent coupling reactions with the substrate to give polymeric coupling products. The molecular weight of these copolymeric products was significantly higher than the molecular weight of products obtained without ABTS. After ultrafiltration, 33% and 21% of the initially applied ABTS could be found in the polymeric product fraction for the substrates guaiacol and erol, respectively, on the basis of nitrogen analysis. When ABTS was added to substrates after full laccase-catalyzed polymerization, the reaction proceeded toward higher molecular weights.

Enzymatic degradation of and bovine serum albumin release from starch-acetate films.

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.

Migration of Contaminants from Milk Tubes and Teat Liners.

The survey tested migration of contaminants from various flexible tubes and teat liners used in milk production. A total of 19 samples were analyzed through sensory evaluation and in migration tests with different simulant liquids. Plasticizers were dissolved in diethyl ether or tetrahydrofuran and analyzed in high performance liquid chromatography (HPLC). The most widely used polyvinyl chloride (PVC) tubes contained 37-40% di-2-ethylhexyl phthalate (DEHP) as plasticizer. In some tubes DEHP was to a great extent replaced by a polymeric plasticizer. DEHP was analyzed with reverse phase HPLC directly from milk in a procedure in which a piece of tube was soaked in milk for 1, 3 and 6 d. Tubes containing polymeric plasticizer lost less DEHP into milk, but relatively more than the tubes plastieized only by DEHP. The evaporation residue for migration tests to water was significantly greater for polyadipate tubes compared with tubes plastieized only by DEHP. The evaporation residue from the water extract of the tubes plastieized by polymeric plasticizer was by mass-speetral analysis found to consist of various hydrolyzation products of polyester from 1,3-butandiol and adipic acid. The results obtained are discussed in the light of toxieologieal problems connected with plasticizers.