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

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.&nbsp

Fasting does not promote a metabolic memory effect in mouse liver

Background: Tissues may respond differently to a particular stimulus if they have been previously exposed to that same stimulus. Here we tested the hypothesis that a strong metabolic stimulus may elicit a memory effect in the liver. To that end, we examined whether prior fasting, which profoundly impacts hepatic nutrient metabolism, may influence the metabolic response to a subsequent fast. Methods: 24 mice were exposed to two 16h fasts over a 8 week period, each time being allowed to return to their normal growth trajectory, while another group of 24 mice was fed ad libitum throughout. Of each group, half of the mice were euthanized after a 16h fast, whereas the other half was euthanized in the ab libitum fed state. Results: An acute fast significantly increased plasma NEFA, glycerol, β-hydroxybutyrate and liver triglycerides, and decreased plasma glucose, triglycerides, and liver glycogen levels. An acute fast also markedly changed the liver transcriptome, upregulating genes involved in fatty acid catabolism and downregulating genes involved in cholesterol synthesis, and majorly impacted the liver metabolome, reducing the levels of numerous amino acids, glycolysis and TCA cycle intermediates, and nucleotides. However, none of the these changes were significantly influenced by prior fasting. The limited number of genes that were significantly altered by prior fasting are likely false positives. Finally, no significant effect was observed of prior fasting on glucose tolerance. Conclusion: We conclude that previous exposure to fasting in mice does not influence the metabolic response to a subsequent fast, thus arguing against the concept of metabolic memory in the hepatic response to fasting

Hepatic ADTRP overexpression does not influence lipid and glucose metabolism

The peroxisome proliferator-activated receptors (PPARs) are a group of transcription factors belonging to the nuclear receptor superfamily. Since most target genes of PPARs are implicated in lipid and glucose metabolism, regulation by PPARs could be used as a screening tool to identify novel genes involved in lipid or glucose metabolism. Here, we identify Adtrp, a serine hydrolase enzyme that was reported to catalyze the hydrolysis of fatty acid esters of hydroxy fatty acids (FAHFAs), as a novel PPAR-regulated gene. Adtrp was significantly upregulated by PPARa activation in mouse primary hepatocytes, liver slices, and whole liver. In addition, Adtrp was upregulated by PPARc activation in 3L3-L1 adipocytes and in white adipose tissue. ChIP-SEQ identified a strong PPAR-binding site in the immediate upstream promoter of the Adtrp gene. Adenoviral-mediated hepatic overexpression of Adtrp in diet-induced obese mice caused a modest increase in plasma nonesterified fatty acids but did not influence diet-induced obesity, liver triglyceride levels, liver lipidomic profiles, liver transcriptomic profiles, plasma cholesterol, triglyceride, glycerol, and glucose levels. Moreover, hepatic Adtrp overexpression did not lead to significant changes in FAHFA levels in plasma or liver and did not influence glucose and insulin tolerance. Finally, hepatic overexpression of Adtrp did not influence liver triglycerides and levels of plasma metabolites after a 24-h fast. Taken together, our data suggest that despite being a PPAR-regulated gene, hepatic Adtrp does not seem to play a major role in lipid and glucose metabolism and does not regulate FAHFA levels

Fasting does not promote a metabolic memory effect in mouse liver

Background: Tissues may respond differently to a particular stimulus if they have been previously exposed to that same stimulus. Here we tested the hypothesis that a strong metabolic stimulus may elicit a memory effect in the liver. To that end, we examined whether prior fasting, which profoundly impacts hepatic nutrient metabolism, may influence the metabolic response to a subsequent fast. Methods: 24 mice were exposed to two 16h fasts over a 8 week period, each time being allowed to return to their normal growth trajectory, while another group of 24 mice was fed ad libitum throughout. Of each group, half of the mice were euthanized after a 16h fast, whereas the other half was euthanized in the ab libitum fed state. Results: An acute fast significantly increased plasma NEFA, glycerol, β-hydroxybutyrate and liver triglycerides, and decreased plasma glucose, triglycerides, and liver glycogen levels. An acute fast also markedly changed the liver transcriptome, upregulating genes involved in fatty acid catabolism and downregulating genes involved in cholesterol synthesis, and majorly impacted the liver metabolome, reducing the levels of numerous amino acids, glycolysis and TCA cycle intermediates, and nucleotides. However, none of the these changes were significantly influenced by prior fasting. The limited number of genes that were significantly altered by prior fasting are likely false positives. Finally, no significant effect was observed of prior fasting on glucose tolerance. Conclusion: We conclude that previous exposure to fasting in mice does not influence the metabolic response to a subsequent fast, thus arguing against the concept of metabolic memory in the hepatic response to fasting

Hepatic ADTRP overexpression does not influence lipid and glucose metabolism

The peroxisome proliferator-activated receptors (PPARs) are a group of transcription factors belonging to the nuclear receptor superfamily. Since most target genes of PPARs are implicated in lipid and glucose metabolism, regulation by PPARs could be used as a screening tool to identify novel genes involved in lipid or glucose metabolism. Here, we identify Adtrp, a serine hydrolase enzyme that was reported to catalyze the hydrolysis of fatty acid esters of hydroxy fatty acids (FAHFAs), as a novel PPAR-regulated gene. Adtrp was significantly upregulated by PPARa activation in mouse primary hepatocytes, liver slices, and whole liver. In addition, Adtrp was upregulated by PPARc activation in 3L3-L1 adipocytes and in white adipose tissue. ChIP-SEQ identified a strong PPAR-binding site in the immediate upstream promoter of the Adtrp gene. Adenoviral-mediated hepatic overexpression of Adtrp in diet-induced obese mice caused a modest increase in plasma nonesterified fatty acids but did not influence diet-induced obesity, liver triglyceride levels, liver lipidomic profiles, liver transcriptomic profiles, plasma cholesterol, triglyceride, glycerol, and glucose levels. Moreover, hepatic Adtrp overexpression did not lead to significant changes in FAHFA levels in plasma or liver and did not influence glucose and insulin tolerance. Finally, hepatic overexpression of Adtrp did not influence liver triglycerides and levels of plasma metabolites after a 24-h fast. Taken together, our data suggest that despite being a PPAR-regulated gene, hepatic Adtrp does not seem to play a major role in lipid and glucose metabolism and does not regulate FAHFA levels

Probing metabolic memory in the hepatic response to fasting

Tissues may respond differently to a particular stimulus if they have been previously exposed to that same stimulus. Here we tested the hypothesis that a strong metabolic stimulus such as fasting may influence the hepatic response to a subsequent fast and thus elicit a memory effect. Overnight fasting in mice significantly increased plasma free fatty acids, glycerol, β-hydroxybutyrate and liver triglycerides, and decreased plasma glucose, plasma triglycerides, and liver glycogen levels. In addition, fasting dramatically changed the liver transcriptome, upregulating genes involved in gluconeogenesis and in uptake, oxidation, storage, and mobilization of fatty acids, and downregulating genes involved in fatty acid synthesis, fatty acid elongation/desaturation, and cholesterol synthesis. Fasting also markedly impacted the liver metabolome, causing a decrease in the levels of numerous amino acids, glycolytic intermediated, TCA cycle intermediates, and nucleotides. However, these fasting-induced changes were unaffected by two previous overnight fasts. Also, no significant effect was observed of prior fasting on glucose tolerance. Finally, analysis of the effect of fasting on the transcriptome in hepatocyte humanized mouse livers indicated modest similarity in gene regulation in mouse and human liver cells. In general, genes involved in metabolic pathways were up- or downregulated to a lesser extent in human liver cells than mouse liver cells. In conclusion, we found that previous exposure to fasting in mice did not influence the hepatic response to a subsequent fast, arguing against the concept of metabolic memory in the liver. Our data provide a useful resource for the study of liver metabolism during fasting

Transcriptomic signature of fasting in adipose tissue

This SuperSeries is composed of the SubSeries listed below. Overall design: Refer to individual Serie

Transcriptomic signature of fasting in murine adipose tissue

Little is known about the impact of fasting on gene regulation in human adipose tissue. Accordingly, the objective of this study was to investigate the effects of fasting on adipose tissue gene expression in humans. To that end, subcutaneous adipose tissue biopsies were collected from volunteers 2h and 26h after consumption of a standardized meal. For comparison, epididymal adipose tissue was collected from C57Bl/6J mice after a 16h fast and in the ab-libitum fed state. Transcriptome analysis was carried out using Affymetrix microarrays. We found that, 1) fasting downregulated numerous metabolic pathways in human adipose tissue, including triglyceride and fatty acid synthesis, glycolysis and glycogen synthesis, TCA cycle, oxidative phosphorylation, mitochondrial translation, and insulin signaling; 2) fasting downregulated genes involved in proteasomal degradation in human adipose tissue; 3) fasting had much less pronounced effects on the adipose tissue transcriptome in humans than mi ce; 4) although major overlap in fasting-induced gene regulation was observed between human and mouse adipose tissue, many genes were differentially regulated in the two species, including genes involved in insulin signaling (PRKAG2, PFKFB3), PPAR signaling (PPARG, ACSL1, HMGCS2, SLC22A5, ACOT1), glycogen metabolism (PCK1, PYGB), and lipid droplets (PLIN1, PNPLA2, CIDEA, CIDEC). In conclusion, although numerous genes and pathways are regulated similarly by fasting in human and mouse adipose tissue, many genes show very distinct responses to fasting in humans and mice. Our data provide a useful resource to study adipose tissue function during fasting. Overall design: Microarray analysis was performed on gonadal adipose tissue in the fed or after a 16 hours fast: Three to four month old C57BL/6 mice were fasted for 16 hours or fed ad libitum. Gonadal white adipose tissue was collected to find changes in gene expression upon fasting in the white adipose tissue

Probing metabolic memory in the hepatic response to fasting

Tissues may respond differently to a particular stimulus if they have been previously exposed to that same stimulus. Here, we tested the hypothesis that a strong metabolic stimulus such as fasting may influence the hepatic response to a subsequent fast and thus elicit a memory effect. Overnight fasting in mice significantly increased plasma free fatty acids, glycerol, b-hydroxybutyrate, and liver triglycerides, and decreased plasma glucose, plasma triglycerides, and liver glycogen levels. In addition, fasting dramatically changed the liver transcriptome, upregulating genes involved in gluconeogenesis and in uptake, oxidation, storage, and mobilization of fatty acids, and downregulating genes involved in fatty acid synthesis, fatty acid elongation/desaturation, and cholesterol synthesis. Fasting also markedly impacted the liver metabolome, causing a decrease in the levels of numerous amino acids, glycolytic-intermediates, TCA cycle intermediates, and nucleotides. However, these fasting-induced changes were unaffected by two previous overnight fasts. Also, no significant effect was observed of prior fasting on glucose tolerance. Finally, analysis of the effect of fasting on the transcriptome in hepatocyte humanized mouse livers indicated modest similarity in gene regulation in mouse and human liver cells. In general, genes involved in metabolic pathways were upregulated or downregulated to a lesser extent in human liver cells than in mouse liver cells. In conclusion, we found that previous exposure to fasting in mice did not influence the hepatic response to a subsequent fast, arguing against the concept of metabolic memory in the liver. Our data provide a useful resource for the study of liver metabolism during fasting

Transcriptomic signature of fasting in human adipose tissue

Little is known about gene regulation by fasting in human adipose tissue. Accordingly, the objec-tive of this study was to investigate the effects of fasting on adipose tissue gene expression in humans. To that end, subcutaneous adipose tissue biopsies were collected from 11 volunteers 2 and 26 h after consumption of a standardized meal. For comparison, epididymal adipose tissue was collected from C57Bl/6J mice in the ab libi-tum-fed state and after a 16 h fast. The timing of sampling adipose tissue roughly corresponds with the near depletion of liver glycogen. Transcriptome analysis was carried out using Affy-metrix microarrays. We found that, 1) fasting downregulated numerous metabolic pathways in human adipose tissue, including triglyceride and fatty acid synthesis, glycolysis and glycogen syn-thesis, TCA cycle, oxidative phosphorylation, mitochondrial trans-lation, and insulin signaling; 2) fasting downregulated genes involved in proteasomal degradation in human adipose tissue; 3) fasting had much less pronounced effects on the adipose tissue transcrip-tome in humans than mice; 4) although major overlap in fasting-induced gene regulation was observed between human and mouse adipose tissue, many genes were differentially regulated in the two species, including genes involved in insulin signaling (PRKAG2, PFKFB3), PPAR signaling (PPARG, ACSL1, HMGCS2, SLC22A5, ACOT1), glycogen metabolism (PCK1, PYGB), and lipid droplets (PLIN1, PNPLA2, CIDEA, CIDEC). In conclusion, although numerous genes and pathways are regulated similarly by fasting in human and mouse adipose tissue, many genes show very distinct responses to fasting in humans and mice. Our data provide a useful resource to study adipose tissue function during fasting