Heli Nygren

Novel Theranostic Opportunities Offered by Characterization of Altered Membrane Lipid Metabolism in Breast Cancer Progression

Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many cancers. However, the precise molecular composition of lipids in tumors remains generally poorly characterized. The aim of the present study was to analyze the global lipid profiles of breast cancer, integrate the results to protein expression, and validate the findings by functional experiments. Comprehensive lipidomics was conducted in 267 human breast tissues using ultraperformance liquid chromatography/ mass spectrometry. The products of de novo fatty acid synthesis incorporated into membrane phospholipids, such as palmitate-containing phosphatidylcholines, were increased in tumors as compared with normal breast tissues. These lipids were associated with cancer progression and patient survival, as their concentration was highest in estrogen receptor-negative and grade 3 tumors. In silico transcriptomics database was utilized in investigating the expression of lipid metabolism related genes in breast cancer, and on the basis of these results, the expression of specific proteins was studied by immunohistochemistry. Immunohistochemical analyses showed that several genes regulating lipid metabolism were highly expressed in clinical breast cancer samples and supported also the lipidomics results. Gene silencing experiments with seven genes [ACACA (acetyl-CoA carboxylase α), ELOVL1 (elongation of very long chain fatty acid-like 1), FASN (fatty acid synthase), INSIG1 (insulin-induced gene 1), SCAP (sterol regulatory element-binding protein cleavage-activating protein), SCD (stearoyl-CoA desaturase), and THRSP (thyroid hormone-responsive protein)] indicated that silencing of multiple lipid metabolism-regulating genes reduced the lipidomic profiles and viability of the breast cancer cells. Taken together, our results imply that phospholipids may have diagnostic potential as well as that modulation of their metabolism may provide therapeutic opportunities in breast cancer treatment.

Liquid Chromatography-Mass Spectrometry (LC-MS)-Based Lipidomics for Studies of Body Fluids and Tissues

In this paper, analytical methodologies for the global profiling of lipids in serum and tissue samples are reported. The sample preparation is based on a modified Folch extraction, and the analysis is carried out with ultrahigh-performance liquid chromatography combined with mass spectrometry (UPLC-MS). For further identification, MS(n) mass spectrometry is carried out utilizing an LTQ-Orbitrap mass spectrometry as the detector. Such a system affords determination of accurate masses and is thus a highly useful tool for lipid identification. The repeatability of the analysis proved to be good, with relative standard errors for spiked samples being between 4.51 and 10.44%. The throughput of the methodology described here is over 100 samples a day.

Prediction of non-alcoholic fatty-liver disease and liver fat content by serum molecular lipids

Aims/hypothesisWe examined whether analysis of lipids by ultra-performance liquid chromatography (UPLC) coupled to MS allows the development of a laboratory test for non-alcoholic fatty-liver disease (NAFLD), and how a lipid-profile biomarker compares with the prediction of NAFLD and liver-fat content based on routinely available clinical and laboratory data.MethodsWe analysed the concentrations of molecular lipids by UPLC-MS in blood samples of 679 well-characterised individuals in whom liver-fat content was measured using proton magnetic resonance spectroscopy (1H-MRS) or liver biopsy. The participants were divided into biomarker-discovery (n = 287) and validation (n = 392) groups to build and validate the diagnostic models, respectively.ResultsIndividuals with NAFLD had increased triacylglycerols with low carbon number and double-bond content while lysophosphatidylcholines and ether phospholipids were diminished in those with NAFLD. A serum-lipid signature comprising three molecular lipids (‘lipid triplet’) was developed to estimate the percentage of liver fat. It had a sensitivity of 69.1% and specificity of 73.8% when applied for diagnosis of NAFLD in the validation series. The usefulness of the lipid triplet was demonstrated in a weight-loss intervention study.Conclusions/interpretationThe liver-fat-biomarker signature based on molecular lipids may provide a non-invasive tool to diagnose NAFLD, in addition to highlighting lipid molecular pathways involved in the disease.

High Density Lipoprotein Structural Changes and Drug Response in Lipidomic Profiles following the Long-Term Fenofibrate Therapy in the FIELD Substudy

In a recent FIELD study the fenofibrate therapy surprisingly failed to achieve significant benefit over placebo in the primary endpoint of coronary heart disease events. Increased levels of atherogenic homocysteine were observed in some patients assigned to fenofibrate therapy but the molecular mechanisms behind this are poorly understood. Herein we investigated HDL lipidomic profiles associated with fenofibrate treatment and the drug-induced Hcy levels in the FIELD substudy. We found that fenofibrate leads to complex HDL compositional changes including increased apoA-II, diminishment of lysophosphatidylcholines and increase of sphingomyelins. Ethanolamine plasmalogens were diminished only in a subgroup of fenofibrate-treated patients with elevated homocysteine levels. Finally we performed molecular dynamics simulations to qualitatively reconstitute HDL particles in silico. We found that increased number of apoA-II excludes neutral lipids from HDL surface and apoA-II is more deeply buried in the lipid matrix than apoA-I. In conclusion, a detailed molecular characterization of HDL may provide surrogates for predictors of drug response and thus help identify the patients who might benefit from fenofibrate treatment.

Cord Serum Lipidome in Prediction of Islet Autoimmunity and Type 1 Diabetes

Previous studies show that children who later progress to type 1 diabetes (T1D) have decreased preautoimmune concentrations of multiple phospholipids as compared with nonprogressors. It is still unclear whether these changes associate with development of β-cell autoimmunity or specifically with clinical T1D. Here, we studied umbilical cord serum lipidome in infants who later developed T1D (N = 33); infants who developed three or four (N = 31) islet autoantibodies, two (N = 31) islet autoantibodies, or one (N = 48) islet autoantibody during the follow-up; and controls (N = 143) matched for sex, HLA-DQB1 genotype, city of birth, and period of birth. The analyses of serum molecular lipids were performed using the established lipidomics platform based on ultra-performance liquid chromatography coupled to mass spectrometry. We found that T1D progressors are characterized by a distinct cord blood lipidomic profile that includes reduced major choline-containing phospholipids, including sphingomyelins and phosphatidylcholines. A molecular signature was developed comprising seven lipids that predicted high risk for progression to T1D with an odds ratio of 5.94 (95% CI, 1.07–17.50). Reduction in choline-containing phospholipids in cord blood therefore is specifically associated with progression to T1D but not with development of β-cell autoimmunity in general.

Increased dihydroceramide/ceramide ratio mediated by defective expression of degs1 impairs adipocyte differentiation and function.

Adipose tissue dysfunction is an important determinant of obesity-associated, lipid-induced metabolic complications. Ceramides are well-known mediators of lipid-induced insulin resistance in peripheral organs such as muscle. DEGS1 is the desaturase catalyzing the last step in the main ceramide biosynthetic pathway. Functional suppression of DEGS1 activity results in substantial changes in ceramide species likely to affect fundamental biological functions such as oxidative stress, cell survival, and proliferation. Here, we show that degs1 expression is specifically decreased in the adipose tissue of obese patients and murine models of genetic and nutritional obesity. Moreover, loss-of-function experiments using pharmacological or genetic ablation of DEGS1 in preadipocytes prevented adipogenesis and decreased lipid accumulation. This was associated with elevated oxidative stress, cellular death, and blockage of the cell cycle. These effects were coupled with increased dihydroceramide content. Finally, we validated in vivo that pharmacological inhibition of DEGS1 impairs adipocyte differentiation. These data identify DEGS1 as a new potential target to restore adipose tissue function and prevent obesity-associated metabolic disturbances.

Interaction between dietary lipids and gut microbiota regulates hepatic cholesterol metabolism

The gut microbiota influences many aspects of host metabolism. We have previously shown that the presence of a gut microbiota remodels lipid composition. Here we investigated how interaction between gut microbiota and dietary lipids regulates lipid composition in the liver and plasma, and gene expression in the liver. Germ-free and conventionally raised mice were fed a lard or fish oil diet for 11 weeks. We performed lipidomics analysis of the liver and serum and microarray analysis of the liver. As expected, most of the variation in the lipidomics dataset was induced by the diet, and abundance of most lipid classes differed between mice fed lard and fish oil. However, the gut microbiota also affected lipid composition. The gut microbiota increased hepatic levels of cholesterol and cholesteryl esters in mice fed lard, but not in mice fed fish oil. Serum levels of cholesterol and cholesteryl esters were not affected by the gut microbiota. Genes encoding enzymes involved in cholesterol biosynthesis were downregulated by the gut microbiota in mice fed lard and were expressed at a low level in mice fed fish oil independent of microbial status. In summary, we show that gut microbiota-induced regulation of hepatic cholesterol metabolism is dependent on dietary lipid composition.

Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells

The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.

Steroid and steroid glucuronide profiles in urine during pregnancy determined by liquid chromatography-electrospray ionization-tandem mass spectrometry

An ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-MS/MS) method was developed for the analysis of steroids and their glucuronides in urine samples. The method provides high sensitivity and fast analysis, as both steroids and their glucuronides can be analyzed directly without hydrolysis or complex sample preparation. The method was applied in profiling of targeted and nontargeted steroids and steroid glucuronides during pregnancy. The concentrations of 11 of 27 targeted steroids and steroid glucuronides and the concentrations of 25 nontargeted steroid glucuronides increased about 10-400 fold during the pregnancy. The concentrations of most of these 36 compounds began to increase in the first days of the pregnancy, increased gradually during the pregnancy, achieved a maximum in late pregnancy, and decreased sharply after delivery. Exceptionally, the concentrations of allopregnanolone and 17-hydroxypregnenolone started to increase later than those of the other steroids. Moreover, the concentrations of E2 glucuronides began to decrease one week before the delivery, in contrast to most of the steroids and steroid glucuronides, whose concentrations dropped sharply during the delivery. Concentrations of 34 compounds decreased noticeably when the subject was on sick leave owing a series of painful contractions. The results suggest that steroids and especially steroid glucuronides may provide a valuable diagnostic tool to follow the course of pregnancy.

Imbalance of plasma amino acids, metabolites and lipids in patients with lysinuric protein intolerance (LPI)

BACKGROUND Lysinuric protein intolerance (LPI [MIM 222700]) is an aminoaciduria with defective transport of cationic amino acids in epithelial cells in the small intestine and proximal kidney tubules due to mutations in the SLC7A7 gene. LPI is characterized by protein malnutrition, failure to thrive and hyperammonemia. Many patients also suffer from combined hyperlipidemia and chronic kidney disease (CKD) with an unknown etiology. METHODS Here, we studied the plasma metabolomes of the Finnish LPI patients (n=26) and healthy control individuals (n=19) using a targeted platform for analysis of amino acids as well as two analytical platforms with comprehensive coverage of molecular lipids and polar metabolites. RESULTS Our results demonstrated that LPI patients have a dichotomy of amino acid profiles, with both decreased essential and increased non-essential amino acids. Altered levels of metabolites participating in pathways such as sugar, energy, amino acid and lipid metabolism were observed. Furthermore, of these metabolites, myo-inositol, threonic acid, 2,5-furandicarboxylic acid, galactaric acid, 4-hydroxyphenylacetic acid, indole-3-acetic acid and beta-aminoisobutyric acid associated significantly (P<0.001) with the CKD status. Lipid analysis showed reduced levels of phosphatidylcholines and elevated levels of triacylglycerols, of which long-chain triacylglycerols associated (P<0.01) with CKD. CONCLUSIONS This study revealed an amino acid imbalance affecting the basic cellular metabolism, disturbances in plasma lipid composition suggesting hepatic steatosis and fibrosis and novel metabolites correlating with CKD in LPI. In addition, the CKD-associated metabolite profile along with increased nitrite plasma levels suggests that LPI may be characterized by increased oxidative stress and apoptosis, altered microbial metabolism in the intestine and uremic toxicity.