Fatty acids are important regulators in the control of mammalian energy homeostasis. They are ingested in the diet but a significant amount are also endogenously produced by de novo lipogenesis. Fatty acid elongation beyond 16 carbons (palmitic acid) can occur to generate very long chain fatty acids (VLCFA), a process that is initiated by the rate-limiting condensation reaction. To date, six mammalian enzymes responsible for this reaction, ELOVL1-6 (Elongation of very long chain fatty acid), have been characterized. All of them exert substrate specificity and tissue-specific gene expression. In this thesis, factors that regulate fatty acid metabolism and, in particular, fatty acid synthesis and elongation will be presented. The enclosed papers discuss issues as to how Elovl3 is regulated in liver and in different adipose depots and what effects ablation of this enzyme causes to lipid homeostasis. Hepatic Elovl3 gene expression followed a circadian rhythm, present exclusively in sexually mature male mice. In contrast to the expression of several other lipogenic genes, Elovl3 gene expression was not affected by fasting or refeeding. Instead, the gene expression was influenced by steroid hormones such as glucocorticoids and sex hormones. Interestingly, despite reduced levels of leptin, Elovl3-ablated mice were shown to be resistant to diet induced weight gain, which seemed to be due to a decreased ratio between energy intake and energy expenditure. This phenotype was more pronounced in female mice. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Thesis (Ph. D.)--University of Lund, 1983. / Includes bibliographical references.

No description available.

One of the cellular responses to temperature fluctuations in plants is the adjustment in the degree of membrane unsaturation. Glycerolipids are major constituents of cellular membranes. In higher plants, glycerolipids are synthesized via two major metabolic pathways compartmentalized in the ER and chloroplast. Adaptive responses in membrane lipids include alterations in fatty acid desaturation, proportional changes in membrane lipids as well as molecular composition of each lipid species. In this study, I systematically explored the significance of glycerolipid pathway balance in temperature induced lipid composition changes in three plant species that have distinctive modes of lipid pathway interactions through a combination of biochemical and molecular approaches including lipidomics and RNA-seq analysis. In Arabidopsis thaliana, a 16:3 plant, low temperature induces an augmented prokaryotic pathway, whereas high temperature enhances the eukaryotic pathway. Atriplex lentiformis reduces its overall lipid desaturation at high temperature and switches lipid phenotype from 16:3 to 18:3 through drastically increasing the contribution of the eukaryotic pathway as well as suppression of the prokaryotic pathway. In sync with the metabolic changes, coordinated expression of glycerolipid pathway genes, as revealed by RNA-seq also occurs. In Triticum aestivum, an 18:3 plant, low temperature leads to a reduced glycerolipid flux from ER to chloroplast. Evidence of differential trafficking of diacylglycerol (DAG) moieties from ER to chloroplast was uncovered in three plant species as another layer of metabolic adaptation under different temperatures. Taken together, this study has established a biochemical basis that highlights the predominance and prevalence of lipid pathway interactions in temperature induced lipid compositional changes.

An individual’s naïve level of response (LR) to ethanol is predictive of their lifetime likelihood to abuse alcohol. LR is heavily genetically influenced, suggesting that the genes responsible for LR may also be central to the development of abuse disorders. Our laboratory uses the model organism C. elegans to investigate the genetic influences on responses to acute ethanol exposure. We recently found that changes in TAG levels can alter LR. From this result we investigated the role of long-chain polyunsaturated fatty acids (LC-PUFAs) as well enzymes involved in lipid modifications of proteins. We found that LC-PUFAs are necessary for acute functional tolerance and that supplementation of eicosapentaenoic acid is able to rescue AFT. We also identified mutations in several palmitoyltransferases, a thioesterase, and elongases that alter AFT. These novel results highlight the importance of fatty acids in the response to ethanol and suggest exciting new potential therapeutic targets.

The primary function of adipose tissue is to store energy in the form of triacylglycerol, which is hydrolyzed to fatty acids to supply other tissues with energy. While insulin promotes the storage of triacylglycerol, catecholamines stimulate its hydrolysis. The development of type II diabetes is strongly associated with obesity, indicating a role of triacylglycerol metabolism in the pathogenesis of diabetes. Caveolae are plasma membrane invaginations found in most cells but are highly abundant in adipocytes. Insulin receptors are localized in caveolae and their function depends on intact caveolae structures. In the present thesis work, mass spectrometry-based methodology allowed identification of a number of new proteins and their posttranslational modifications in caveolae of human adipocytes. Variable N-terminal acetylation and phosphorylation of caveolin-1α and caveolin-1β were identified, which might regulate the function of caveolae. The transcription regulator protein PTRF was identified as the major caveolae associated protein. Specific proteolytic modifications of PTRF at the cytosolic surface of caveolae and phosphorylation on nine serine and one threonine residues were identified. Moreover, insulin induced translocation of PTRF from the plasma membrane to the nucleus. PTRF was previously shown to regulate the activity of both RNA polymerase I and polymerase II, thus a role of PTRF in mediating the anabolic action of insulin on protein synthesis and gene transcription is proposed. PTRF was also involved in an extranuclear function in the hormonal regulation of triacylglycerol metabolism in caveolae. PTRF was colocalized with the triacylglycerol regulator proteins perilipin and hormone-sensitive lipase (HSL) in the triacylglycerol-synthesizing caveolae subclass. We showed that, while perilipin was translocated to the plasma membrane, both PTRF and HSL were translocated from the plasma membrane to the cytosol as a complex in response to insulin. The perilipin recruited to the plasma membrane was highly threonine phosphorylated. By mass spectrometry, three phosphorylated threonine residues were identified and were located in an acidic domain in the lipid droplet targeting domain of perilipin. The insulin-induced recruitment of perilipin to the plasma membrane might, therefore be phosphorylation-dependent. Isoproterenol, which stimulates hydrolysis of triacylglycerol, induced a complete depletion of perilipin B from the plasma membrane, suggesting a function of perilipin B to protect newly synthesized triacylglycerol in caveolae from being hydrolyzed by HSL. The location of PTRF and HSL was not affected by isoproterenol, indicating that insulin is acting against a default presence of PTRF and HSL in caveolae. Taken together, this thesis expands our knowledge about caveolae and provided valuable information about their involvement in novel roles, particularly in the hormonal regulation of triacylglycerol metabolism.

This thesis is a series of investigations into the regulation of skeletal muscle mitochondrial metabolism. Novel regulatory mechanisms regarding mitochondrial fatty acid oxidation are continually being identified and alterations in skeletal muscle mitochondrial metabolism have been implicated in the pathogenesis of type II diabetes (T2DM). Therefore, advancing our basic understanding of mitochondrial regulatory processes is required to provide insight into the progression of T2DM. In study one, the utilization of knockout mice for the putative mitochondrial fatty acid transport protein FAT/CD36, showed that mitochondrial FAT/CD36 plays a functional role in mitochondrial long chain fatty acid (LCFA) oxidation. Specifically, FAT/CD36 was found to be located on the outer mitochondrial membrane (OMM) upstream of acyl-CoA synthetase. In study two, it was observed that in rat muscle, malonyl-CoA (M-CoA) inhibition kinetics of carnitine palmitoyltransferase I (CPT-I) display a more physiological IC50 in permeabilzed muscle fibre bundles (PmFB) compared to isolated mitochondria. These data suggest that the cytoskeleton may have a role in regulating M-CoA inhibition. Additionally, a significant effect of LCFA-CoA on M-CoA inhibition kinetics was observed. These data indicate that M-CoA content does not need to decrease to promote an increase in CPT-I flux. Finally, in a model of T2DM (ZDF rat), submaximal ADP-stimulated respiration rates and the content of adenine nucleotide translocase 2 (ANT2) content were depressed compared to lean control animals. Resveratrol treatment in ZDF rats recovered these declines concomitantly with improving insulin-stimulated skeletal muscle glucose uptake and the cellular redox state. A number of novel findings are presented, specifically, 1) a functional role for mitochondrial FAT/CD36 in mitochondrial LCFA oxidation was confirmed and the topology of this protein along the OMM is expanded upon, 2) M-CoA inhibition kinetics of CPT-I were re-evaluated in PmFB and a regulatory role of LCFA-CoA on M-CoA inhibition kinetics is established, and 3) submaximal ADP-stimulated respiration rates and ANT2 content are depressed in the ZDF rat and resveratrol supplementation prevents these decrements.

The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.

Obesity, type 2 diabetes, and cardiovascular diseases are major problems in today's society, causing millions of deaths every year. One of the main risk factors for these diseases is a dysregulation of the fatty acid metabolism, where the balance between release and uptake of fatty acids is disturbed. Thus, understanding how fatty acid metabolism works is of great importance in the battle against these diseases. The human fatty acid release and uptake can be unraveled by measuring the difference in metabolite concentrations between an artery before the adipose tissue and a vein draining the tissue. Such measurements are called arterio-venous. However, due to the complexity of the fatty acid mechanisms, the resulting measurements alone are not enough to understand all of the involved reactions governing the metabolism. One analytical tool to decipher such complex mechanisms is mathematical modeling. A few mathematical models have previously used arterio-venous data of the fatty acid metabolism, but none of the previous models describe a full day including several meals and nightly fast. In this project, I combine mathematical modeling and arterio-venous data to investigate the mechanisms of fatty acid metabolism during three consecutive meals and fasting. The resulting mathematical model can explain arterio-venous data of free fatty acids, triglycerides, and glycerol. The model predictions show that re-esterification of monacylglycerides, a mechanism that has not been considered before when analyzing arterio-venous data, is of importance to be able to accurately describe the fatty acid metabolism. Additionally, the model predicts that there is a hormonal regulation during the night. Finally, it is shown that many of the previous simple calculations used to approximate metabolic reactions do not capture the desired reactions but instead calculate more complex properties, while the use of the model allows for a more detailed analysis separating all of the different reaction rates. These results give new insights into the complex mechanisms of fatty acid metabolism and provide a new tool to analyze arterio-venous data more comprehensively. In the future, this can lead to a better understanding of metabolic diseases such as obesity, type 2 diabetes, and cardiovascular diseases.

No description available.