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| 2. |
- Fernandez, Celine, et al.
(författare)
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Disturbed cholesterol homeostasis in hormone-sensitive lipase-null mice.
- 2008
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Ingår i: American journal of physiology. Endocrinology and metabolism. - : American Physiological Society. - 0193-1849 .- 1522-1555. ; 295:4
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Tidskriftsartikel (refereegranskat)abstract
- Transcriptomics analysis revealed that genes involved in hepatic de novo cholesterol synthesis were downregulated in fed HSL-null mice that had been on a high-fat diet (HFD) for 6 mo. This finding prompted a further analysis of cholesterol metabolism in HSL-null mice, which was performed in fed and 16-h-fasted mice on a normal chow diet (ND) or HFD regimen. Plasma cholesterol was elevated in HSL-null mice, in all tested conditions, as a result of cholesterol enrichment of HDL and VLDL. Hepatic esterified cholesterol content and ATP-binding cassette transporter A1 (ABCA1) mRNA and protein levels were increased in HSL-null mice regardless of the dietary regimen. Unsaturated fatty acid composition of hepatic triglycerides was modified in fasted HSL-null mice on ND and HFD. The increased ABCA1 expression had no major effect on cholesterol efflux from HSL-null mouse hepatocytes. Taken together, the results of this study suggest that HSL plays a critical role in the hydrolysis of cytosolic cholesteryl esters and that increased levels of hepatic cholesteryl esters, due to lack of action of HSL in the liver, are the main mechanism underlying the imbalance in cholesterol metabolism in HSL-null mice.
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| 3. |
- Fernandez, Celine, et al.
(författare)
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Hormone-sensitive lipase is necessary for normal mobilization of lipids during sub-maximal exercise.
- 2008
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Ingår i: American Journal of Physiology: Endocrinology and Metabolism. - : American Physiological Society. - 1522-1555 .- 0193-1849. ; 295, s. 179-186
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Tidskriftsartikel (refereegranskat)abstract
- For the working muscle there are a number of fuels available for oxidative metabolism, including glycogen, glucose and non-esterified fatty acids. Non-esterified fatty acids originate from lipolysis in white adipose tissue, from hydrolysis of VLDL-triglycerides or from hydrolysis of intramyocellular triglyceride stores. A key enzyme in the mobilization of fatty acids from intracellular lipid stores is hormone-sensitive lipase (HSL). The aim of the present study was to investigate the metabolic response of HSL-null mice challenged with exercise or fasting and to examine if other lipases are able to fully compensate for the lack of HSL. The results showed that HSL-null mice have reduced capacity to perform aerobic exercise. The liver glycogen stores were more rapidly depleted in HSL-null mice during treadmill exercise and HSL-null mice had reduced plasma concentrations of both glycerol and non-esterified fatty acids after exercise and fasting, respectively. The data support the hypothesis that in the absence of HSL mice are not able to respond to an exercise challenge with increased mobilization of the lipid stores. Consequently, the impact of the lipid sparing effect on liver glycogen will be reduced in the HSL-null mice, resulting in faster depletion of this energy source, contributing to the decreased endurance during sub-maximal exercise. Key words: Treadmill exercise, lipid metabolism, glycogen, skeletal muscle, liver.
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| 4. |
- Fernandez, Celine, et al.
(författare)
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Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13).
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:1, s. 400-411
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Tidskriftsartikel (refereegranskat)abstract
- Metabolites generated from fuel metabolism in pancreatic beta-cells control exocytosis of insulin, a process which fails in type 2 diabetes. To identify and quantify these metabolites, global and unbiased analysis of cellular metabolism is required. To this end, polar metabolites, extracted from the clonal 832/13 beta-cell line cultured at 2.8 and 16.7 mM glucose for 48 h, were derivatized followed by identification and quantification, using gas chromatography (GC) and mass spectrometry (MS). After culture at 16.7 mM glucose for 48 h, 832/13 beta-cells exhibited a phenotype reminiscent of glucotoxicity with decreased content and secretion of insulin. The metabolomic analysis revealed alterations in the levels of 7 metabolites derived from glycolysis, the TCA cycle and pentose phosphate shunt, and 4 amino acids. Principal component analysis of the metabolite data showed two clusters, corresponding to the cells cultured at 2.8 and 16.7 mM glucose, respectively. Concurrent changes in protein expression were analyzed by 2-D gel electrophoresis followed by LC-MS/MS. The identities of 86 spots corresponding to 75 unique proteins that were significantly different in 832/13 beta-cells cultured at 16.7 mM glucose were established. Only 5 of these were found to be metabolic enzymes that could be involved in the metabolomic alterations observed. Anticipated changes in metabolite levels in cells exposed to increased glucose were observed, while changes in enzyme levels were much less profound. This suggests that substrate availability, allosteric regulation, and/or post-translational modifications are more important determinants of metabolite levels than enzyme expression at the protein level.
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| 5. |
- Fernandez, Céline, et al.
(författare)
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Omics Analyses Reveal a Potential Link between Hormone-Sensitive Lipase and Polyamine Metabolism.
- 2009
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8, s. 5008-5019
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization from lipid stores, is expressed in the liver and decreased hepatic insulin sensitivity has been reported in our HSL null mouse model. Here, an integrated approach, comprising transcriptomics and proteomics together with targeted metabolite analysis, was used to investigate the liver phenotype of HSL null mice. Oligonucleotide microarray analysis revealed altered expression of genes involved in lipid and polyamine metabolism in HSL null mice compared with wild-type mice and in genes controlling the immune system in mice on high-fat diet versus mice on normal diet. Two-dimensional gel electrophoresis followed by MS and/or MS/MS allowed identification of 52 and 22 unique proteins differentially regulated according to the genotype and diet, respectively. Changes were observed mainly for proteins related to metabolism, including several proteins involved in polyamine metabolism or exhibiting methyl transferase activity. Despite the coordinated changes in mRNA and protein levels in polyamine pathways, no significant differences in levels of key polyamine metabolites were detected between the two genotypes. This study identifies a link between HSL and polyamine metabolism, which deserves further attention in view of the emerging data suggesting that disturbances in polyamine metabolism may affect insulin sensitivity. The present work also describes a limited correlation between mRNA, protein and metabolite levels, thus, underscoring the importance of integrated approaches.
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| 6. |
- Fernandez, Celine
(författare)
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Omics Techniques Applied to Diabetes Research - Focus on HSL-Null Mice and Clonal β-Cells
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diabetes mellitus is a disease characterized by increased blood glucose levels. For overt type 2 diabetes to develop, both insulin action and insulin secretion must be perturbed. In the work presented in this thesis, two models were analyzed: hormone-sensitive lipase (HSL) null mice and a pancreatic clonal β-cell line to study the mechanisms underlying insulin resistance and insulin secretion perturbations, respectively. To achieve this, techniques allowing a global analysis of the transcriptome, proteome and metabolome were used. HSL is best known as the enzyme hydrolyzing acylglycerides stored in adipose tissue. But HSL has broad substrate specificity and is widely expressed elsewhere than in adipose tissue. Its role in non-adipose tissues is not completely understood, but the phenotype revealed by the characterization of several independently generated HSL-null mouse lines during the recent years suggests that HSL has several functions in addition to its role in adipocyte lipolysis. The role of HSL in the liver was studied in this thesis. More specifically, the liver phenotype of HSL-null mice was investigated at the transcriptome (Paper I and IV) and proteome (Paper IV) levels. The obtained results didn’t allow the obvious identification of possible mechanisms behind the hepatic insulin resistance observed in our HSL-null mouse strain. However, our results show that HSL plays an important role as a cholesteryl ester hydrolase in the liver and that HSL influences overall cholesterol homeostasis by indirectly controlling hepatic HDL-cholesterol clearance. We also demonstrated the importance of a cross-talk between white adipose tissue and liver that regulates cholesterol homeostasis via the type of non-esterified fatty acids (NEFA) released. Moreover, changes in expression of proteins involved in polyamine metabolism were observed in the liver of HSL-null mice, which could be responsible for the increased liver weight characterizing HSL-null mice. The physiological response of HSL-null mice to aerobic treadmill exercise was also investigated (Paper II). HSL was shown to play an important role during aerobic exercise in controlling the mobilization of lipid stores from white adipose tissue, a function which cannot be fully compensated by any other acylglyceride lipases. Proteome and metabolome analyses were performed in Paper III to study glucose-stimulated insulin secretion in a β-cell line cultured in presence of normal or toxic glucose concentrations. A metabolite fingerprint which is characteristic for β-cells cultured at high glucose concentrations was obtained.
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| 7. |
- Krogh, Morten, et al.
(författare)
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A probabilistic treatment of the missing spot problem in 2D gel electrophoresis experiments
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:8, s. 3335-3343
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Tidskriftsartikel (refereegranskat)abstract
- Two-dimensional SIDS-PAGE gel electrophoresis using post-run staining is widely used to measure the abundances of thousands of protein spots simultaneously. Usually, the protein abundances of two or more biological groups are compared using biological and technical replicates. After gel separation and staining, the spots are detected, spot volumes are quantified, and spots are matched across gels. There are almost always many missing values in the resulting data set. The missing values arise either because the corresponding proteins have very low abundances (or are absent) or because of experimental errors such as incomplete/over focusing in the first dimension or varying run times in the second dimension as well as faulty spot detection and matching. In this study, we show that the probability for a spot to be missing can be modeled by a logistic regression function of the logarithm of the volume. Furthermore, we present an algorithm that takes a set of gels with technical and biological replicates as input and estimates the average protein abundances in the biological groups from the number of missing spots and measured volumes of the present spots using a maximum likelihood approach. Confidence intervals for abundances and p-values for differential expression between two groups are calculated using bootstrap sampling. The algorithm is compared to two standard approaches, one that discards missing values and one that sets all missing values to zero. We have evaluated this approach in two different gel data sets of different biological origin. An F-program, implementing the algorithm, is freely available at httP://bioinfo.thep.lu.se/MissingValues2Dgels.html.
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| 8. |
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| 9. |
- Nilsson, Lars, et al.
(författare)
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Competitive adsorption of proteins from total hen egg yolk during emulsification
- 2007
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 55:16, s. 6746-6753
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Tidskriftsartikel (refereegranskat)abstract
- In this article, the competitive adsorption of egg yolk proteins at oil/water interfaces during emulsification is studied. By using two-dimensional polyacrylamide electrophoresis and mass spectrometry, it was possible to characterize and identify adsorbing and non-adsorbing protein species. The egg yolk contains proteins with a wide range of molecular weights and pI. Lipoproteins adsorbed selectively throughout the pH range investigated. It is suggested that selectivity is determined by the average hydrophobic and hydrophilic domain lengths in the protein sequences where long average hydrophobic domain lengths result in high affinity for the interface and thus strong preferential adsorption.
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| 10. |
- Nilsson, Lars, et al.
(författare)
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Competitive adsorption of water soluble plasma proteins from egg yolk at the oil/water interface
- 2006
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 54:18, s. 6881-6887
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Tidskriftsartikel (refereegranskat)abstract
- Water soluble plasma proteins were fractionated from hen's egg yolk, and the molecular weight and pI of the most abundant protein species were characterized with gel electrophoresis. The proteins were identified by mass spectrometry. The protein fraction was used to produce oil-in-water emulsions, both at various protein concentrations and at various pH values, and the surface load was determined through serum depletion. The competitive adsorption was studied through the determination of nonadsorbing species with gel electrophoresis. The results show that it was possible to form an oil-in-water emulsion for which droplet size and maximum surface load depended on the protein concentration and pH. Serum albumin and YGP40 adsorbed selectively at the oil/water interface throughout the pH range investigated, and for albumin the selectivity increased close to its pI. It is suggested that this selective adsorption is due to long hydrophobic stretches in the polypeptide chain, which are present in the selectively adsorbing species but absent in less adsorbing species.
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