Can we ENERGISE our metabolic health? : a deeper look into players of lipid metabolism and the regulation of fasting and cold-induced browning

Samenvatting

Over the past decades, an immense pandemic of obesity has been developing across the world, in part due to an overabundance of food that is available 24/7. While in ancient history a major need existed to store spare energy for times when food was scarce, the energy-conserving and -storing mechanisms that humans evolved with can now be considered counterproductive, as they interfere with the maintenance of a healthy homeostatic state. Chronic overfeeding results in the well-known diseases of affluence such as obesity, non-alcoholic fatty liver disease, and type 2 diabetes mellitus. These conditions are rooted in disturbances in energy metabolism related to chronic overconsumption of foods.

Energy metabolism is carefully regulated at multiple levels via the coordinated action of thousands of genes and proteins. An important group of transcription factors involved in the regulation of many pathways involved in energy metabolism are the peroxisome proliferator-activated receptors (PPARs). These nuclear receptors play a pivotal role in different aspects of glucose and lipid metabolism and inflammation. Three PPAR isoforms have been identified: PPARα (Nrc1), PPARγ (Nrc3) and PPARδ (Nrc2). PPARs are involved in many metabolic pathways via their capacity to activate transcription of a large number of target genes. In order to get a better understanding of the regulation of lipid metabolism, in the first part of this thesis we aimed to look at novel putative target genes of PPAR. By using “regulation via PPAR” as a screening tool to identify novel genes involved in lipid and glucose metabolism, we identified Androgen Dependent TFPI Regulating Protein (ADTRP) (chapter 2) and Transmembrane P24 Trafficking Protein 5 (TMED5) (chapter 3) as genes of high interest. ADTRP encodes a serine hydrolase enzyme that was reported to catalyse the hydrolysis of fatty acid esters of hydroxy fatty acids (FAHFAs). FAHFAs have recently been identified as a potential insulin-sensitizing class of lipids. Based on the current literature and the data presented in this thesis, we could not support a major role for ADTRP in glucose or lipid metabolism in the liver since neither hepatic overexpression nor deficiency of ADTRP resulted in a clear metabolic phenotype in the liver. Additionally, upon overexpression of ADTRP, no changes in FAHFA concentrations were found in liver or plasma, questioning the role of ADTRP in FAHFA hydrolysis in the liver. For TMED5, we could not detect alterations in metabolic phenotype following overexpression of Tmed5 in mice fed a high fat diet or subjected to fasting. Taken together, in this thesis we clearly show that ADTRP and TMED5 are targets of PPAR. However, based on the current data, we were not able to support a major role of hepatic ADTRP or TMED5 in glucose or lipid metabolism. In the second part of this thesis, we had a deeper look into different stressors of metabolism. First in chapter 4, we studied the role of PPARα during cold-induced  browning. Exposing male mice for 10 days to 5 degrees compared to a thermoneutral environment resulted in increased food intake and a decrease in adipose tissue weight independent of PPARα. Additionally, also browning occurred to a similar extent in both genotypes.. With this we showed that PPARα is dispensable for cold-induced browning, contrary to the literature showing a diminished thermogenic response in PPARα-/- mice upon β3-adrenergic receptor activation. Another important stressor of metabolism is fasting. During fasting, stored energy becomes available in order for the organism to survive. Two major organs involved in the response to fasting are the liver and the adipose tissue. In the adipose tissue, which is the principal energy depot, fasting activates intracellular lipolysis, thereby releasing free fatty acids and glycerol into the circulation. Simultaneously, fasting represses extracellular lipolysis, leading to reduced uptake and storage of circulating triglycerides. Although the general response to fasting in different mammals is very similar, we carefully studied the transcriptional response to fasting in human adipose tissue and compared these data to the fasting response of mouse adipose tissue in chapter 5. A large number of metabolic pathways were commonly downregulated in mouse and human adipose tissue upon fasting, including triglyceride and fatty acid synthesis, glycolysis and glycogen synthesis, TCA cycle, oxidative phosphorylation, mitochondrial translation, and insulin signaling, even though the magnitude of the effect was much smaller in humans compared to mice. However, we also showed that many genes have a very distinct response to fasting in humans as compared to mice. These differentially regulated genes include genes involved in insulin signaling, PPAR signaling, glycogen metabolism, and lipid droplets. With this data we provide a useful resource for the study of the response to fasting in human adipose tissue and at the same time raise awareness for the need for caution when extrapolating findings from mice to humans. The hepatic fasting response, which is mainly driven by PPARα, has been studied extensively over the past decades. In line with the relatively novel idea of innate immune memory, where macrophages are believed to have an altered response to a previously encountered inflammatory compound such as LPS, we looked into a possible memory effect of fasting in the liver in chapter 6. However, our data do not provide evidence in favor of a lasting footprint of fasting on liver gene expression in mice. We found that previous exposure to fasting did not influence the metabolic phenotype of mice and did not influence the liver transcriptome and metabolome. Since we were the first to study the effect of an episode of fasting on the hepatic levels of numerous polar metabolites, including amino acids, other organic acids, and nucleotides, we do provide a useful resource for the study of liver metabolism during fasting.

In conclusion, with this thesis we aimed to find novel genes or pathways involved in the regulation of lipid and glucose metabolism, with the premise that these genes and pathways may be suitable candidates for therapeutic targeting. Our studies provide important new insights into the regulation of metabolism in liver and adipose tissue in response to cold, fasting, and PPARα activation.