Self-organized metabotyping of obese individuals identifies clusters responding differently to bariatric surgery.

Weight loss through bariatric surgery is efficient for treatment or prevention of obesity related diseases such as type 2 diabetes and cardiovascular disease. Long term weight loss response does, however, vary among patients undergoing surgery. Thus, it is difficult to identify predictive markers while most obese individuals have one or more comorbidities. To overcome such challenges, an in-depth multiple omics analyses including fasting peripheral plasma metabolome, fecal metagenome as well as liver, jejunum, and adipose tissue transcriptome were performed for 106 individuals undergoing bariatric surgery. Machine leaning was applied to explore the metabolic differences in individuals and evaluate if metabolism-based patients' stratification is related to their weight loss responses to bariatric surgery. Using Self-Organizing Maps (SOMs) to analyze the plasma metabolome, we identified five distinct metabotypes, which were differentially enriched for KEGG pathways related to immune functions, fatty acid metabolism, protein-signaling, and obesity pathogenesis. The gut metagenome of the most heavily medicated metabotypes, treated simultaneously for multiple cardiometabolic comorbidities, was significantly enriched in Prevotella and Lactobacillus species. This unbiased stratification into SOM-defined metabotypes identified signatures for each metabolic phenotype and we found that the different metabotypes respond differently to bariatric surgery in terms of weight loss after 12 months. An integrative framework that utilizes SOMs and omics integration was developed for stratifying a heterogeneous bariatric surgery cohort. The multiple omics datasets described in this study reveal that the metabotypes are characterized by a concrete metabolic status and different responses in weight loss and adipose tissue reduction over time. Our study thus opens a path to enable patient stratification and hereby allow for improved clinical treatments.

A systems biology approach to study non-alcoholic fatty liver (NAFL) in women with obesity.

Non-alcoholic fatty liver disease (NAFLD) is now the most frequent global chronic liver disease. Individuals with NAFLD exhibited an increased risk of all-cause mortality driven by extrahepatic cancers and liver and cardiovascular disease. Once the disease is established, women have a higher risk of disease progression and worse outcome. It is therefore critical to deepen the current knowledge on the pathophysiology of NAFLD in women. Here, we used a systems biology approach to investigate the contribution of different organs to this disease. We analyzed transcriptomics profiles of liver and adipose tissues, fecal metagenomes, and plasma metabolomes of 55 women with and without NAFLD. We observed differences in metabolites, expression of human genes, and gut microbial features between the groups and revealed that there is substantial crosstalk between these different omics sets. Multi-omics analysis of individuals with NAFLD may provide novel strategies to study the pathophysiology of NAFLD in humans.

Maternal cecal microbiota transfer rescues early-life antibiotic-induced enhancement of type 1 diabetes in mice.

Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury.

Gut microbial metabolites as multi-kingdom intermediates.

The gut microbiota contributes to host physiology through the production of a myriad of metabolites. These metabolites exert their effects within the host as signalling molecules and substrates for metabolic reactions. Although the study of host-microbiota interactions remains challenging due to the high degree of crosstalk both within and between kingdoms, metabolite-focused research has identified multiple actionable microbial targets that are relevant for host health. Metabolites, as the functional output of combined host and microorganism interactions, provide a snapshot in time of an extraordinarily complex multi-organism system. Although substantial work remains towards understanding host-microbiota interactions and the underlying mechanisms, we review the current state of knowledge for each of the major classes of microbial metabolites with emphasis on clinical and translational research implications. We provide an overview of methodologies available for measurement of microbial metabolites, and in addition to discussion of key challenges, we provide a potential framework for integration of discovery-based metabolite studies with mechanistic work. Finally, we highlight examples in the literature where this approach has led to substantial progress in understanding host-microbiota interactions.

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model.

Humans with metabolic and inflammatory diseases frequently harbour lower levels of butyrate-producing bacteria in their gut. However, it is not known whether variation in the levels of these organisms is causally linked with disease development and whether diet modifies the impact of these bacteria on health. Here we show that a prominent gut-associated butyrate-producing bacterial genus (Roseburia) is inversely correlated with atherosclerotic lesion development in a genetically diverse mouse population. We use germ-free apolipoprotein E-deficient mice colonized with synthetic microbial communities that differ in their capacity to generate butyrate to demonstrate that Roseburia intestinalis interacts with dietary plant polysaccharides to: impact gene expression in the intestine, directing metabolism away from glycolysis and toward fatty acid utilization; lower systemic inflammation; and ameliorate atherosclerosis. Furthermore, intestinal administration of butyrate reduces endotoxaemia and atherosclerosis development. Together, our results illustrate how modifiable diet-by-microbiota interactions impact cardiovascular disease, and suggest that interventions aimed at increasing the representation of butyrate-producing bacteria may provide protection against atherosclerosis.

Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.

The early-life intestinal microbiota plays a key role in shaping host immune system development. We found that a single early-life antibiotic course (1PAT) accelerated type 1 diabetes (T1D) development in male NOD mice. The single course had deep and persistent effects on the intestinal microbiome, leading to altered cecal, hepatic, and serum metabolites. The exposure elicited sex-specific effects on chromatin states in the ileum and liver and perturbed ileal gene expression, altering normal maturational patterns. The global signature changes included specific genes controlling both innate and adaptive immunity. Microbiome analysis revealed four taxa each that potentially protect against or accelerate T1D onset, that were linked in a network model to specific differences in ileal gene expression. This simplified animal model reveals multiple potential pathways to understand pathogenesis by which early-life gut microbiome perturbations alter a global suite of intestinal responses, contributing to the accelerated and enhanced T1D development.

Metabolic programming of the epigenome: host and gut microbial metabolite interactions with host chromatin.

The mammalian gut microbiota has been linked to host developmental, immunologic, and metabolic outcomes. This collection of trillions of microbes inhabits the gut and produces a myriad of metabolites, which are measurable in host circulation and contribute to the pathogenesis of human diseases. The link between endogenous metabolite availability and chromatin regulation is a well-established and active area of investigation; however, whether microbial metabolites can elicit similar effects is less understood. In this review, we focus on seminal and recent research that establishes chromatin regulatory roles for both endogenous and microbial metabolites. We also highlight key physiologic and disease settings where microbial metabolite-host chromatin interactions have been established and/or may be pertinent.

Chemical signaling between gut microbiota and host chromatin: What is your gut really saying?

Mammals and their gut microbial communities share extensive and tightly coordinated co-metabolism of dietary substrates. A large number of microbial metabolites have been detected in host circulation and tissues and, in many cases, are linked to host metabolic, developmental, and immunological states. The presence of these metabolites in host tissues intersects with regulation of the host's epigenetic machinery. Although it is established that the host's epigenetic machinery is sensitive to levels of endogenous metabolites, the roles for microbial metabolites in epigenetic regulation are just beginning to be elucidated. This review focuses on eukaryotic chromatin regulation by endogenous and gut microbial metabolites and how these regulatory events may impact host developmental and metabolic phenotypes.

Diet-Microbiota Interactions Mediate Global Epigenetic Programming in Multiple Host Tissues.

Histone-modifying enzymes regulate transcription and are sensitive to availability of endogenous small-molecule metabolites, allowing chromatin to respond to changes in environment. The gut microbiota produces a myriad of metabolites that affect host physiology and susceptibility to disease; however, the underlying molecular events remain largely unknown. Here we demonstrate that microbial colonization regulates global histone acetylation and methylation in multiple host tissues in a diet-dependent manner: consumption of a "Western-type" diet prevents many of the microbiota-dependent chromatin changes that occur in a polysaccharide-rich diet. Finally, we demonstrate that supplementation of germ-free mice with short-chain fatty acids, major products of gut bacterial fermentation, is sufficient to recapitulate chromatin modification states and transcriptional responses associated with colonization. These findings have profound implications for understanding the complex functional interactions between diet, gut microbiota, and host health.

Reader domain specificity and lysine demethylase-4 family function.

The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitro demethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis.

Iron Deprivation Induces Transcriptional Regulation of Mitochondrial Biogenesis.

Mitochondria are essential organelles that adapt to stress and environmental changes. Among the nutrient signals that affect mitochondrial form and function is iron, whose depletion initiates a rapid and reversible decrease in mitochondrial biogenesis through unclear means. Here we demonstrate that, unlike the canonical iron-induced alterations to transcript stability, loss of iron dampens the transcription of genes encoding mitochondrial proteins with no change to transcript half-life. Using mass spectrometry, we demonstrate that these transcriptional changes are accompanied by dynamic alterations to histone acetylation and methylation levels that are largely reversible upon readministration of iron. Moreover, histone deacetylase inhibition abrogates the decreased histone acetylation observed upon iron deprivation and restores normal transcript levels at genes encoding mitochondrial proteins. Collectively, we demonstrate that deprivation of an essential nutrient induces transcriptional repression of organellar biogenesis involving epigenetic alterations.

Loss of SIRT3 Provides Growth Advantage for B Cell Malignancies.

B cell malignancies comprise a diverse group of cancers that proliferate in lymph nodes, bone marrow, and peripheral blood. SIRT3 (sirtuin 3) is the major deacetylase within the mitochondrial matrix that promotes aerobic metabolism and controls reactive oxygen species (ROS) by deacetylating and activating isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2). There is controversy as to whether SIRT3 acts as an oncogene or a tumor suppressor, and here we investigated its role in B cell malignancies. In mantle cell lymphoma patient samples, we found that lower SIRT3 protein expression was associated with worse overall survival. Further, SIRT3 protein expression was reduced in chronic lymphocytic leukemia primary samples and malignant B cell lines compared to primary B cells from healthy donors. This lower level of expression correlated with hyperacetylation of IDH2 and SOD2 mitochondrial proteins, lowered enzymatic activities, and higher ROS levels. Overexpression of SIRT3 decreased proliferation and diminished the Warburg-like phenotype in SIRT3-deficient cell lines, and this effect is largely dependent on deacetylation of IDH2 and SOD2. Lastly, depletion of SIRT3 from malignant B cell lines resulted in greater susceptibility to treatment with an ROS scavenger but did not result in greater sensitivity to inhibition of the hypoxia-inducible factor-1α pathway, suggesting that loss of SIRT3 increases proliferation via ROS-dependent but hypoxia-inducible factor-1α-independent mechanisms. Our study suggests that SIRT3 acts as a tumor suppressor in B cell malignancies, and activating the SIRT3 pathway might represent a novel therapeutic approach for treating B cell malignancies.

Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry.

Histone post-translational modifications (PTMs) are important regulators of chromatin structure and gene expression. Quantitative analysis of histone PTMs by mass spectrometry remains extremely challenging due to the complex and combinatorial nature of histone PTMs. The most commonly used mass spectrometry-based method for high-throughput histone PTM analysis is data-dependent acquisition (DDA). However, stochastic precursor selection and dependence on MS1 ions for quantification impede comprehensive interrogation of histone PTM states using DDA methods. To overcome these limitations, we utilized a data-independent acquisition (DIA) workflow that provides superior run-to-run consistency and postacquisition flexibility in comparison to DDA methods. In addition, we developed a novel DIA-based methodology to quantify isobaric, co-eluting histone peptides that lack unique MS2 transitions. Our method enabled deconvolution and quantification of histone PTMs that are otherwise refractory to quantitation, including the heavily acetylated tail of histone H4. Using this workflow, we investigated the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) on the global histone PTM state of human breast cancer MCF7 cells. A total of 62 unique histone PTMs were quantified, revealing novel SAHA-induced changes in acetylation and methylation of histones H3 and H4.

Metabolic regulation of histone post-translational modifications.

Histone post-translational modifications regulate transcription and other DNA-templated functions. This process is dynamically regulated by specific modifying enzymes whose activities require metabolites that either serve as cosubstrates or act as activators/inhibitors. Therefore, metabolism can influence histone modification by changing local concentrations of key metabolites. Physiologically, the epigenetic response to metabolism is important for nutrient sensing and environment adaption. In pathologic states, the connection between metabolism and histone modification mediates epigenetic abnormality in complex disease. In this review, we summarize recent studies of the molecular mechanisms involved in metabolic regulation of histone modifications and discuss their biological significance.

Tcf19 is a novel islet factor necessary for proliferation and survival in the INS-1 ß-cell line.

Recently, a novel type 1 diabetes association locus was identified at human chromosome 6p31.3, and transcription factor 19 (TCF19) is a likely causal gene. Little is known about Tcf19, and we now show that it plays a role in both proliferation and apoptosis in insulinoma cells. Tcf19 is expressed in mouse and human islets, with increasing mRNA expression in nondiabetic obesity. The expression of Tcf19 is correlated with ß-cell mass expansion, suggesting that it may be a transcriptional regulator of ß-cell mass. Increasing proliferation and decreasing apoptotic cell death are two strategies to increase pancreatic ß-cell mass and prevent or delay diabetes. siRNA-mediated knockdown of Tcf19 in the INS-1 insulinoma cell line, a ß-cell model, results in a decrease in proliferation and an increase in apoptosis. There was a significant reduction in the expression of numerous cell cycle genes from the late G1 phase through the M phase, and cells were arrested at the G1/S checkpoint. We also observed increased apoptosis and susceptibility to endoplasmic reticulum (ER) stress after Tcf19 knockdown. There was a reduction in expression of genes important for the maintenance of ER homeostasis (Bip, p58(IPK), Edem1, and calreticulin) and an increase in proapoptotic genes (Bim, Bid, Nix, Gadd34, and Pdia2). Therefore, Tcf19 is necessary for both proliferation and survival and is a novel regulator of these pathways.

FoxM1 is up-regulated by obesity and stimulates beta-cell proliferation.

beta-Cell mass expansion is one mechanism by which obese animals compensate for insulin resistance and prevent diabetes. FoxM1 is a transcription factor that can regulate the expression of multiple cell cycle genes and is necessary for the maintenance of adult beta-cell mass, beta-cell proliferation, and glucose homeostasis. We hypothesized that FoxM1 is up-regulated by nondiabetic obesity and initiates a transcriptional program leading to beta-cell proliferation. We performed gene expression analysis on islets from the nondiabetic C57BL/6 Leptin(ob/ob) mouse, the diabetic BTBR Leptin(ob/ob) mouse, and an F2 Leptin(ob/ob) population derived from these strains. We identified obesity-driven coordinated up-regulation of islet Foxm1 and its target genes in the nondiabetic strain, correlating with beta-cell mass expansion and proliferation. This up-regulation was absent in the diabetic strain. In the F2 Leptin(ob/ob) population, increased expression of Foxm1 and its target genes segregated with higher insulin and lower glucose levels. We next studied the effects of FOXM1b overexpression on isolated mouse and human islets. We found that FoxM1 stimulated mouse and human beta-cell proliferation by activating many cell cycle phases. We asked whether FOXM1 expression is also responsive to obesity in human islets by collecting RNA from human islet donors (body mass index range: 24-51). We found that the expression of FOXM1 and its target genes is positively correlated with body mass index. Our data suggest that beta-cell proliferation occurs in adult obese humans in an attempt to expand beta-cell mass to compensate for insulin resistance, and that the FoxM1 transcriptional program plays a key role in this process.