How do we differentiate not demonise - Is there a role for healthier processed foods in an age of food insecurity? Proceedings of a roundtable event.

The 'ultra-processed food' (UPF) concept, with classification of foods by 'level of processing' rather than nutrient profiles, and its relationship with health outcomes, is currently a topic of debate among academics and increasingly referred to in the media. The British Nutrition Foundation convened a virtual roundtable on 6th July 2022 to gather views on the use of the term (and current definitions of) UPF for public health messaging, seeking to establish areas of consensus and disagreement and identify topics for further research. A small group of invited expert stakeholders attended, including representatives from academia, policy, behavioural science, communications, health, food science, retail and consumer interests. Participants' discussions clustered into cogent themes which included: problems with the use of definitions for UPF, the lack of causal evidence and defined mechanisms linking processing per se with poor health outcomes, and advice that may result in consumer confusion. There was agreement that many foods classified as UPF are high in fat, sugars and/or salt and public health messages should continue to focus on reducing these in the diet since it is unclear whether reported associations between high intakes of UPF and poor health reflect poorer dietary patterns (defined by nutrient intakes), and nutrient-health relationships are well established. Examples of misalignment were also highlighted (i.e. some foods are classified as UPF yet recommended in food-based dietary guidelines [featuring in healthy dietary patterns]). This raises challenges for consumer communication around UPF. Concern was also expressed about potential unintended consequences, particularly for vulnerable groups, where advice to avoid UPF could create stigma and guilt due to lack of time or facilities to prepare and cook meals from scratch. It could also impact on nutrient intakes, as some foods classified as UPF represent more affordable sources of important nutrients (e.g. packaged wholemeal bread). Discordance between the concept of UPF and current strategies to improve public health, such as reformulation, was also discussed. The group concluded that the use of the concept of UPF in UK policy (e.g. dietary guidelines) would be unhelpful at present. Overall, participants felt that it was more important to focus on providing practical advice around selection of healthier processed foods and making healthier foods more accessible rather than promoting the avoidance of UPF. The latter may act to demonise all foods classified as UPF by current definitions, including some affordable nutrient-dense foods.

The time is now: Achieving FH paediatric screening across Europe - The Prague Declaration.

Familial hypercholesterolaemia (FH) is the most common inherited metabolic disorder characterized by high cholesterol and if left untreated leads to premature cardiovascular disease, such as heart attacks. Treatment that begins early in life, particularly in childhood, is highly efficacious in preventing cardiovascular disease and cost-effective, thus early detection of FH is crucial. However, in Europe, less than 10% of people living with FH are diagnosed and even less receive life-saving treatment. The Prague Declaration is a call to action for national and European Union policymakers and decision-makers and a result of the Czech EU Presidency meeting on FH Paediatric Screening (early detection of inherited high cholesterol) at the Czech Senate in Prague on 6th September 2022. It builds on a considerable body of evidence which was discussed at the Technical Meeting under the auspices of the Slovenian EU Presidency in October 2021. The Prague meeting addressed the outstanding barriers to the systematic implementation of FH paediatric screening across Europe. In this article, we present the key points from the Prague meeting and concrete actions needed to move forward.

"Even We Are Confused": A Thematic Analysis of Professionals' Perceptions of Processed Foods and Challenges for Communication.

Processed foods are increasingly under the spotlight since the development of classification systems based on proxies for food processing. Published critical reviews and commentaries suggest different views among professional disciplines about the definition and classification of processed food. There is a need to further understand perspectives of professionals on the conceptualisation of processed food and the agreements and disagreements among experts, to encourage interdisciplinary dialogue and aid communication to the public. The aim of this research was to elicit views and understandings of professionals on processed food, their perceptions of lay people's perceptions of the same, and their perspectives on the challenges of communicating about processed foods to the public. The online discussion groups brought together a range of professionals (n = 27), covering the fields of nutrition, food technology, policy making, industry, and civil society, mixed in 5 heterogenous groups. Through thematic analysis the following themes relating to the conceptualisation of processed food and challenges for communication were identified: (1) Broad concepts that need differentiation; (2) Disagreements on scope and degree of processing; (3) The role of food processing within the food system: the challenges in framing risks and benefits; and (4) The challenge of different perspectives and interests for risk communication. Throughout the discussions blurred lines in the characterisation of processing, processed foods, and unhealthy foods were observed. Participants agreed that consensus is important, but difficult. Participants identified a need for further interdisciplinary dialogue, including public engagement, to break down the observed issues, and work towards a mutual understanding and develop clear communication messages.

Food-Based Dietary Guidelines - development of a conceptual framework for future Food-Based Dietary Guidelines in Europe: report of a Federation of European Nutrition Societies Task-Force Workshop in Copenhagen, 12-13 March 2018.

Identifying a need for developing a conceptual framework for the future development of Food-Based Dietary Guidelines (FBDG) in Europe, The Federation of European Nutrition Sciences established a Task Force for this purpose. A workshop was held with the specific objective to discuss the various dimensions considered as particularly relevant. Existing frameworks for FBDG were discussed, and presentations from various countries illustrated not only several commonalities but also a high degree of heterogeneity in the guidelines from different countries. Environmental aspects were considered in several countries, and dimensions like food safety, dietary habits and preparation were included in others. The workshop provided an overview of the use of FBDG - both in developing front-of-pack nutrition labels and for reformulation and innovation. The European FBDG dimensions were described with examples from the close connection between FBDG and European Union (EU) policies and activities and from the compilation of a database of national FBDG. Also, the challenges with communication of FBDG were discussed. Considering the current scientific basis and the experiences from several countries, the Task Force discussed the various dimensions of developing FBDG and concluded that environmental aspects should be included in the future conceptual framework for FBDG. A change in terminology to sustainable FDBG (SFBDG) could reflect this. The Task Force concluded that further work needs to be done exploring current practice, existing methodologies and the future prospects for incorporating other relevant dimensions into a future Federation of European Nutrition Societies conceptual framework for SFBDG in Europe and working groups were formed to address that.

Effect of Hyperglycemia on Gene Expression during Early Organogenesis in Mice.

BACKGROUND: Cardiovascular and neural malformations are common sequels of diabetic pregnancies, but the underlying molecular mechanisms remain unknown. We hypothesized that maternal hyperglycemia would affect the embryos most shortly after the glucose-sensitive time window at embryonic day (ED) 7.5 in mice. METHODS: Mice were made diabetic with streptozotocin, treated with slow-release insulin implants and mated. Pregnancy aggravated hyperglycemia. Gene expression profiles were determined in ED8.5 and ED9.5 embryos from diabetic and control mice using Serial Analysis of Gene Expression and deep sequencing. RESULTS: Maternal hyperglycemia induced differential regulation of 1,024 and 2,148 unique functional genes on ED8.5 and ED9.5, respectively, mostly in downward direction. Pathway analysis showed that ED8.5 embryos suffered mainly from impaired cell proliferation, and ED9.5 embryos from impaired cytoskeletal remodeling and oxidative phosphorylation (all P ≤ E-5). A query of the Mouse Genome Database showed that 20-25% of the differentially expressed genes were caused by cardiovascular and/or neural malformations, if deficient. Despite high glucose levels in embryos with maternal hyperglycemia and a ~150-fold higher rate of ATP production from glycolysis than from oxidative phosphorylation on ED9.5, ATP production from both glycolysis and oxidative phosphorylation was reduced to ~70% of controls, implying a shortage of energy production in hyperglycemic embryos. CONCLUSION: Maternal hyperglycemia suppressed cell proliferation during gastrulation and cytoskeletal remodeling during early organogenesis. 20-25% of the genes that were differentially regulated by hyperglycemia were associated with relevant congenital malformations. Unexpectedly, maternal hyperglycemia also endangered the energy supply of the embryo by suppressing its glycolytic capacity.

Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance.

BACKGROUND: The nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to modulate the inflammatory response of macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice. RESULTS: In line with previous observations, SDF-1α (CXCL12) was among the most upregulated genes in Nur77-deficient BMM and we demonstrated that Nur77 binds directly to the SDF-1α promoter, resulting in inhibition of SDF-1α expression. The cytokine receptor CX3CR1 was strongly downregulated in Nur77-KO BMM, implying involvement of Nur77 in macrophage tolerance. Ingenuity pathway analyses (IPA) to identify canonical pathways regulation and gene set enrichment analyses (GSEA) revealed a potential role for Nur77 in extracellular matrix homeostasis. Nur77-deficiency increased the collagen content of macrophage extracellular matrix through enhanced expression of several collagen subtypes and diminished matrix metalloproteinase (MMP)-9 activity. IPA upstream regulator analyses discerned the small GTPase Rac1 as a novel regulator of Nur77-mediated gene expression. We identified an inhibitory feedback loop with increased Rac1 activity in Nur77-KO BMM, which may explain the augmented phagocytic activity of these cells. Finally, we predict multiple chronic inflammatory diseases to be influenced by macrophage Nur77 expression. GSEA and IPA associated Nur77 to osteoarthritis, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, and allergic airway inflammatory diseases. CONCLUSIONS: Altogether these data identify Nur77 as a modulator of macrophage function and an interesting target to treat chronic inflammatory disease.

LIM-only protein FHL2 is a positive regulator of liver X receptors in smooth muscle cells involved in lipid homeostasis.

The LIM-only protein FHL2 is expressed in smooth muscle cells (SMCs) and inhibits SMC-rich-lesion formation. To further elucidate the role of FHL2 in SMCs, we compared the transcriptomes of SMCs derived from wild-type (WT) and FHL2 knockout (KO) mice. This revealed that in addition to the previously recognized involvement of FHL2 in SMC proliferation, the cholesterol synthesis and liver X receptor (LXR) pathways are altered in the absence of FHL2. Using coimmunoprecipitation experiments, we found that FHL2 interacts with the two LXR isoforms, LXRα and LXRß. Furthermore, FHL2 strongly enhances transcriptional activity of LXR element (LXRE)-containing reporter constructs. Chromatin immunoprecipitation (ChIP) experiments on the ABCG1 promoter revealed that FHL2 enhances the association of LXRß with DNA. In line with these observations, we observed reduced basal transcriptional LXR activity in FHL2-KO SMCs compared to WT SMCs. This was also reflected in reduced expression of LXR target genes in intact aorta and aortic SMCs of FHL2-KO mice. Functionally, the absence of FHL2 resulted in attenuated cholesterol efflux to both ApoA-1 and high-density lipoprotein (HDL), in agreement with reduced LXR signaling. Collectively, our findings demonstrate that FHL2 is a transcriptional coactivator of LXRs and points toward FHL2 being an important determinant of cholesterol metabolism in SMCs.

Integrated visualization of a multi-omics study of starvation in mouse intestine.

Our understanding of complex biological processes can be enhanced by combining different kinds of high-throughput experimental data, but the use of incompatible identifiers makes data integration a challenge. We aimed to improve methods for integrating and visualizing different types of omics data. To validate these methods, we applied them to two previous studies on starvation in mice, one using proteomics and the other using transcriptomics technology. We extended the PathVisio software with new plugins to link proteins, transcripts and pathways. A low overall correlation between proteome and transcriptome data was detected (Spearman rank correlation: 0.21). At the level of individual genes, correlation was highly variable. Many mRNA/protein pairs, such as fructose biphosphate aldolase B and ATP Synthase, show good correlation. For other pairs, such as ferritin and elongation factor 2, an interesting effect is observed, where mRNA and protein levels change in opposite directions, suggesting they are not primarily regulated at the transcriptional level. We used pathway diagrams to visualize the integrated datasets and found it encouraging that transcriptomics and proteomics data supported each other at the pathway level. Visualization of the integrated dataset on pathways led to new observations on gene-regulation in the response of the gut to starvation. Our methods are generic and can be applied to any multi-omics study. The PathVisio software can be obtained at http://www.pathvisio.org. Supplemental data are available at http://www.bigcat.unimaas.nl/data/jib-supplemental/ , including instructions on reproducing the pathway visualizations of this manuscript.

Fasting reduces liver fibrosis in a mouse model for chronic cholangiopathies.

Chronic cholangiopathies often lead to fibrosis, as a result of a perpetuated wound healing response, characterized by increased inflammation and excessive deposition of proteins of the extracellular matrix. Our previous studies have shown that food deprivation suppresses the immune response, which led us to postulate its beneficial effects on pathology in liver fibrosis driven by portal inflammation. We investigated the consequences of fasting on liver fibrosis in Abcb4(-/-) mice that spontaneously develop it due to a lack of phospholipids in bile. The effect of up to 48h of food deprivation was studied by gene expression profiling, (immuno)histochemistry, and biochemical assessments of biliary output, and hepatic and plasma lipid composition. In contrast to increased biliary output in the wild type counterparts, bile composition in Abcb4(-/-) mice remained unchanged with fasting and did not influence the attenuation of fibrosis. Markers of inflammation, however, dramatically decreased in livers of Abcb4(-/-) mice already after 12h of fasting. Reduced presence of activated hepatic stellate cells and actively increased tissue remodeling further propelled a decrease in parenchymal fibrosis in fasting. This study is the first to show that food deprivation positively influences liver pathology in a fibrotic mouse model for chronic cholangiopathies, opening a door for new strategies to improve liver regeneration in chronic disease.

Overexpression of insulin like growth factor binding protein 5 reduces liver fibrosis in chronic cholangiopathy.

The ATP-binding cassette, sub-family B member 4 knock-out mouse (Abcb4(-/-)) is a relevant model for chronic cholangiopathy in man. Due to the lack of this P-glycoprotein in the canalicular membrane of hepatocytes, the secretion of phospholipids into bile is absent, resulting in increased bile toxicity. Expression of insulin like growth factor binding protein 5 (Igfbp5) increases in time in the livers of these mice. It is unclear whether this induction is a consequence of or plays a role in the progression of liver pathology. The aim of this study was therefore to investigate the effect of IGFBP5 induction on the progression of liver fibrosis caused by chronic cholangiopathy. IGFBP5 and, as a control, green fluorescent protein were overexpressed in the hepatocytes of Abcb4(-/-) mice, using an adeno-associated viral vector (AAV). Progression of liver fibrosis was studied 3, 6, and 12 weeks after vector injection by analyzing serum parameters, collagen deposition, expression of pro-fibrotic genes, inflammation and oxidative stress. A single administration of the AAV vectors provided prolonged expression of IGFBP5 and GFP in the livers of Abcb4(-/-) mice. Compared to GFP control, fractional liver weight, extracellular matrix deposition and amount of activated hepatic stellate cells significantly decreased in IGFBP5 overexpressing mice even 12 weeks after treatment. This effect was not due to a change in bile composition, but driven by reduced inflammation, oxidative stress, and proliferation. Overexpression of IGFBP5 seems to have a protective effect on liver pathology in this model for chronic cholangiopathy.

Interorgan coordination of the murine adaptive response to fasting.

Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0-72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted "carbohydrate-lipid-protein" succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.

Unexpected effects of fasting on murine lipid homeostasis--transcriptomic and lipid profiling.

BACKGROUND & AIMS: Starvation induces massive perturbations in metabolic pathways involved in energy metabolism, but its effect on the metabolism of lipids, particularly cholesterol, is little understood. METHODS: A comparative genomic analysis of the gut and the liver in response to fasting was performed, with intestinal perfusion and lipid profiling of the plasma, bile, liver, intestinal tissue, perfusate, and faeces in FVB mice. RESULTS: The expression profiles suggested increased cholesterol trafficking in the liver and decreased trafficking in the small intestine. Plasma cholesterol concentrations significantly increased, and triglycerides decreased in fasting. Surprisingly, in prolonged fasting, the biliary bile salt and lipid output rates increased, with increased hepatic and intestinal lipid turnover, and enhanced trans-intestinal cholesterol excretion. In contrast, faecal sterol loss declined sharply. To investigate whether the increased biliary phospholipid secretion could nourish the intestinal epithelium, we studied the histology of the small intestines upon fasting in multidrug resistant protein 2 deficient mice with scarce biliary phospholipids. Their adaptive biliary response to fasting was lost, while the shortage of biliary phospholipids strongly induced apoptosis and proliferation in the small intestine and increased the number of mucin-producing cells. CONCLUSION: Even with no dietary fat, lipid levels remain remarkably constant in the murine liver and intestines during prolonged fasting. The biliary system, always assumed to be coupled to the postprandial response, shows a paradoxical increase in activity. We hypothesise that biliary lipids are mobilised to supply the enterocytes with luminal fuel and to stabilise transport systems in the intestine for ensuring a rapid recovery when the food supply resumes.

Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells.

BACKGROUND: Expression of insulin-like growth factor binding protein 5 (IGFBP5) is strongly induced upon activation of hepatic stellate cells and their transdifferentiation into myofibroblasts in vitro. This was confirmed in vivo in an animal model of liver fibrosis. Since IGFBP5 has been shown to promote fibrosis in other tissues, the aim of this study was to investigate its role in the progression of liver fibrosis. METHODS: The effect of IGFBP5 was studied in LX2 cells, a model for partially activated hepatic stellate cells, and in human primary liver myofibroblasts. IGFBP5 signalling was modulated by the addition of recombinant protein, by lentiviral overexpression, and by siRNA mediated silencing. Furthermore, the addition of IGF1 and silencing of the IGF1R was used to investigate the role of the IGF-axis in IGFBP5 mediated effects. RESULTS: IGFBP5 enhanced the survival of LX2 cells and myofibroblasts via a >50% suppression of apoptosis. This effect of IGFBP5 was not modulated by the addition of IGF1, nor by silencing of the IGF1R. Additionally, IGFBP5 was able to enhance the expression of established pro-fibrotic markers, such as collagen Ialpha1, TIMP1 and MMP1. CONCLUSION: IGFBP5 enhances the survival of (partially) activated hepatic stellate cells and myofibroblasts by lowering apoptosis via an IGF1-independent mechanism, and enhances the expression of profibrotic genes. Its lowered expression may, therefore, reduce the progression of liver fibrosis.

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.

Modulation of glycosphingolipid metabolism significantly improves hepatic insulin sensitivity and reverses hepatic steatosis in mice.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, and type 2 diabetes. The hyperinsulinemia that occurs as a consequence of insulin resistance is thought to be an important contributor to the development of fatty liver. We have shown that the iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM), an inhibitor of the enzyme glucosylceramide synthase, is a potent enhancer of insulin signaling in rodent models for insulin resistance and type 2 diabetes. The present study was designed to assess the impact of AMP-DNM on insulin levels, liver triglyceride synthesis, and gene expression profile. Treatment of ob/ob mice with AMP-DNM restored insulin signaling in the liver, corrected blood glucose values to levels found in lean mice, and decreased insulin concentration. The expression of sterol regulatory element-binding protein 1c target genes involved in fatty acid synthesis normalized. AMP-DNM treatment significantly reduced liver to body weight ratio and reversed hepatic steatosis, comprising fat as well as inflammatory markers. In addition, AMP-DNM treatment corrected to a large extent the gene expression profile of ob/ob mice livers toward the profile of lean mice. CONCLUSION: Pharmacological lowering of glycosphingolipids with the iminosugar AMP-DNM is a promising approach to restore insulin signaling and improve glucose homeostasis as well as hepatic steatosis.

Reduction of glycosphingolipid biosynthesis stimulates biliary lipid secretion in mice.

UNLABELLED: Recent reports indicate that glycosphingolipids play an important role in regulation of carbohydrate metabolism. We have shown that the iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM), an inhibitor of the enzyme glucosylceramide synthase, is a potent enhancer of insulin signaling in rodent models for insulin resistance and type 2 diabetes. In this study, we determined whether AMP-DNM also affects lipid homeostasis and, in particular, the reverse cholesterol transport pathway. Treatment of C57BL/6J mice with AMP-DNM for 5 weeks decreased plasma levels of triglycerides and cholesterol by 35%, whereas neutral sterol excretion increased twofold. Secretion of biliary lipid also increased twofold, which resulted in a similar rise in bile flow. This effect was not due to altered expression levels or kinetics of the various export pumps involved in bile formation. However, the bile salt pool size increased and the expression of Cyp7A1 was up-regulated. In vitro experiments using HepG2 hepatoma cell line revealed this to be due to inhibition of fibroblast growth factor-19 (FGF19)-mediated suppression of Cyp7A1 via the FGF receptor. CONCLUSION: Pharmacological modulation of glycosphingolipid metabolism showed surprising effects on lipid homeostasis in C57BL/6J mice. Upon administration of 100 mg AMP-DNM/kg body weight/day, plasma cholesterol and triglyceride levels decreased, biliary lipid secretion doubled and also the endpoint of reverse cholesterol transport, neutral sterol excretion, doubled.

The transcriptomic signature of fasting murine liver.

BACKGROUND: The contribution of individual organs to the whole-body adaptive response to fasting has not been established. Hence, gene-expression profiling, pathway, network and gene-set enrichment analysis and immunohistochemistry were carried out on mouse liver after 0, 12, 24 and 72 hours of fasting. RESULTS: Liver wet weight had declined approximately 44, approximately 5, approximately 11 and approximately 10% per day after 12, 24, 48 and 72 hours of fasting, respectively. Liver structure and metabolic zonation were preserved. Supervised hierarchical clustering showed separation between the fed, 12-24 h-fasted and 72 h-fasted conditions. Expression profiling and pathway analysis revealed that genes involved in amino-acid, lipid, carbohydrate and energy metabolism responded most significantly to fasting, that the response peaked at 24 hours, and had largely abated by 72 hours. The strong induction of the urea cycle, in combination with increased expression of enzymes of the tricarboxylic-acid cycle and oxidative phosphorylation, indicated a strong stimulation of amino-acid oxidation peaking at 24 hours. At this time point, fatty-acid oxidation and ketone-body formation were also induced. The induction of genes involved in the unfolded-protein response underscored the cell stress due to enhanced energy metabolism. The continuous high expression of enzymes of the urea cycle, malate-aspartate shuttle, and the gluconeogenic enzyme Pepck and the re-appearance of glycogen in the pericentral hepatocytes indicate that amino-acid oxidation yields to glucose and glycogen synthesis during prolonged fasting. CONCLUSION: The changes in liver gene expression during fasting indicate that, in the mouse, energy production predominates during early fasting and that glucose production and glycogen synthesis become predominant during prolonged fasting.

Fasting induces a biphasic adaptive metabolic response in murine small intestine.

BACKGROUND: The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. RESULTS: Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine, and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed. CONCLUSION: The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover.

Starvation induces phase-specific changes in the proteome of mouse small intestine.

Food deprivation results in metabolic, structural, and functional changes in the small intestine that influences gut mucosal integrity, epithelial cell proliferation, mucin synthesis, and other processes. The underlying mechanisms are still unclear, which lead to the study of molecular effects of short-term and long-term starvation in the intestine of mice. A comparative proteomics approach, combining two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, was used to identify intestinal proteins whose expression is changed under different starvation conditions (0, 12, 24, and 72 h). In total, the expression levels of 80 protein spots changed significantly between the different groups. The results demonstrate that after 12 h of starvation, mainly proteins involved in glycolysis and energy metabolism show decreased expression levels. Starvation for 24 h results in a down-regulation of proteins involved in protein synthesis and amino acid metabolism. Simultaneously, proteins with a protective role, e.g., reg I and II, glutathione peroxidase 3, and carbonic anhydrase 3, are clearly up-regulated. The last starvation phase (72 h) is characterized by increased ezrin expression, which may enhance villus morphogenesis critical for survival. Together, these results provide novel insights in the intestinal starvation response and may contribute to improved nutritional support during conditions characterized by malnutrition.