Monoallelic intragenic POU3F2 variants lead to neurodevelopmental delay and hyperphagic obesity, confirming the gene's candidacy in 6q16.1 deletions.

While common obesity accounts for an increasing global health burden, its monogenic forms have taught us underlying mechanisms via more than 20 single-gene disorders. Among these, the most common mechanism is central nervous system dysregulation of food intake and satiety, often accompanied by neurodevelopmental delay (NDD) and autism spectrum disorder. In a family with syndromic obesity, we identified a monoallelic truncating variant in POU3F2 (alias BRN2) encoding a neural transcription factor, which has previously been suggested as a driver of obesity and NDD in individuals with the 6q16.1 deletion. In an international collaboration, we identified ultra-rare truncating and missense variants in another ten individuals sharing autism spectrum disorder, NDD, and adolescent-onset obesity. Affected individuals presented with low-to-normal birth weight and infantile feeding difficulties but developed insulin resistance and hyperphagia during childhood. Except for a variant leading to early truncation of the protein, identified variants showed adequate nuclear translocation but overall disturbed DNA-binding ability and promotor activation. In a cohort with common non-syndromic obesity, we independently observed a negative correlation of POU3F2 gene expression with BMI, suggesting a role beyond monogenic obesity. In summary, we propose deleterious intragenic variants of POU3F2 to cause transcriptional dysregulation associated with hyperphagic obesity of adolescent onset with variable NDD.

Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion through Myeloid Cell Retinoic Acid Production.

Pancreatic-islet inflammation contributes to the failure of ß cell insulin secretion during obesity and type 2 diabetes. However, little is known about the nature and function of resident immune cells in this context or in homeostasis. Here we show that interleukin (IL)-33 was produced by islet mesenchymal cells and enhanced by a diabetes milieu (glucose, IL-1ß, and palmitate). IL-33 promoted ß cell function through islet-resident group 2 innate lymphoid cells (ILC2s) that elicited retinoic acid (RA)-producing capacities in macrophages and dendritic cells via the secretion of IL-13 and colony-stimulating factor 2. In turn, local RA signaled to the ß cells to increase insulin secretion. This IL-33-ILC2 axis was activated after acute ß cell stress but was defective during chronic obesity. Accordingly, IL-33 injections rescued islet function in obese mice. Our findings provide evidence that an immunometabolic crosstalk between islet-derived IL-33, ILC2s, and myeloid cells fosters insulin secretion.

snRNA-seq reveals a subpopulation of adipocytes that regulates thermogenesis.

Adipose tissue is usually classified on the basis of its function as white, brown or beige (brite)1. It is an important regulator of systemic metabolism, as shown by the fact that dysfunctional adipose tissue in obesity leads to a variety of secondary metabolic complications2,3. In addition, adipose tissue functions as a signalling hub that regulates systemic metabolism through paracrine and endocrine signals4. Here we use single-nucleus RNA-sequencing (snRNA-seq) analysis in mice and humans to characterize adipocyte heterogeneity. We identify a rare subpopulation of adipocytes in mice that increases in abundance at higher temperatures, and we show that this subpopulation regulates the activity of neighbouring adipocytes through acetate-mediated modulation of their thermogenic capacity. Human adipose tissue contains higher numbers of cells of this subpopulation, which could explain the lower thermogenic activity of human compared to mouse adipose tissue and suggests that targeting this pathway could be used to restore thermogenic activity.

Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle.

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium.

The obesity-linked human lncRNA AATBC stimulates mitochondrial function in adipocytes.

Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, thermogenic adipocytes are linked to cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this plasticity could help improving metabolism. Here, we show that the lncRNA Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Comparing transcriptional profiles of human adipose tissues and cultured adipocytes we discovered that AATBC was enriched in thermogenic conditions. Using primary and immortalized human adipocytes we found that AATBC enhanced the thermogenic phenotype, which was linked to increased respiration and a more fragmented mitochondrial network. Expression of AATBC in adipose tissue of mice led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, BMI, and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.

A stromal cell population that inhibits adipogenesis in mammalian fat depots.

Adipocyte development and differentiation have an important role in the aetiology of obesity and its co-morbidities1,2. Although multiple studies have investigated the adipogenic stem and precursor cells that give rise to mature adipocytes3-14, our understanding of their in vivo origin and properties is incomplete2,15,16. This is partially due to the highly heterogeneous and unstructured nature of adipose tissue depots17, which has proven difficult to molecularly dissect using classical approaches such as fluorescence-activated cell sorting and Cre-lox lines based on candidate marker genes16,18. Here, using the resolving power of single-cell transcriptomics19 in a mouse model, we reveal distinct subpopulations of adipose stem and precursor cells in the stromal vascular fraction of subcutaneous adipose tissue. We identify one of these subpopulations as CD142+ adipogenesis-regulatory cells, which can suppress adipocyte formation in vivo and in vitro in a paracrine manner. We show that adipogenesis-regulatory cells are refractory to adipogenesis and that they are functionally conserved in humans. Our findings point to a potentially critical role for adipogenesis-regulatory cells in modulating adipose tissue plasticity, which is linked to metabolic control, differential insulin sensitivity and type 2 diabetes.

Local acetate inhibits brown adipose tissue function.

Brown adipose tissue has been extensively studied in the last decade for its potential to counteract the obesity pandemic. However, the paracrine regulation within brown tissue is largely unknown. Here, we show that local acetate directly inhibits brown fat thermogenesis, without changing acetate levels in the circulation. We demonstrate that modulating acetate within brown tissue at physiological levels blunts its function and systemically decreases energy expenditure. Using a series of transcriptomic analyses, we identified genes related to the tricarboxylic acid cycle and brown adipocyte formation, which are down-regulated upon local acetate administration. Overall, these findings demonstrate that local acetate inhibits brown fat function.

FGF-2-dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis.

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2-dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.

Enantioselective Total Syntheses of Cassane Furanoditerpenoids and Their Stimulation of Cellular Respiration in Brown Adipocytes.

We report the first and enantioselective total syntheses of (+)-1-deacetylcaesalmin C, (+)-δ-caesalpin, (+)-norcaesalpinin MC, and (+)-norcaesalpinin P. Salient features of the synthetic strategy are an exo-selective intramolecular Diels-Alder reaction of a furanoquinone monoketal and subsequent chemoselective reduction of the resulting pentacyclic furfuryl ketal, furnishing a keystone intermediate. The latter enables access to the collection of natural products through implementation of stereoselective oxidations. Having accessed the cassane furanoditerpenoids, we unveil previously unknown bioactivity: (+)-1-deacetylcaesalmin C stimulates respiration in brown adipocytes, which has been suggested to play a central role in treatment of obesity.

Adipose-derived circulating miRNAs regulate gene expression in other tissues.

Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine.

The Role of IL-13 and IL-4 in Adipose Tissue Fibrosis.

White adipose tissue (WAT) fibrosis, characterized by an excess of extracellular (ECM) matrix components, is strongly associated with WAT inflammation and dysfunction due to obesity. Interleukin (IL)-13 and IL-4 were recently identified as critical mediators in the pathogenesis of fibrotic diseases. However, their role in WAT fibrosis is still ill-defined. We therefore established an ex vivo WAT organotypic culture system and demonstrated an upregulation of fibrosis-related genes and an increase of α-smooth muscle actin (αSMA) and fibronectin abundance upon dose-dependent stimulation with IL-13/IL-4. These fibrotic effects were lost in WAT lacking il4ra, which encodes for the underlying receptor controlling this process. Adipose tissue macrophages were found to play a key role in mediating IL-13/IL-4 effects in WAT fibrosis as their depletion through clodronate dramatically decreased the fibrotic phenotype. IL-4-induced WAT fibrosis was partly confirmed in mice injected intraperitoneally with IL-4. Furthermore, gene correlation analyses of human WAT samples revealed a strong positive correlation of fibrosis markers with IL-13/IL-4 receptors, whereas IL13 and IL4 correlations failed to confirm this association. In conclusion, IL-13 and IL-4 can induce WAT fibrosis ex vivo and partly in vivo, but their role in human WAT remains to be further elucidated.

Obesity Is Associated with Distorted Proteoglycan Expression in Adipose Tissue.

Proteoglycans are central components of the extracellular matrix (ECM) and binding partners for inflammatory chemokines. Morphological differences in the ECM and increased inflammation are prominent features of the white adipose tissues in patients with obesity. The impact of obesity and weight loss on the expression of specific proteoglycans in adipose tissue is not well known. This study aimed to investigate the relationship between adiposity and proteoglycan expression. We analyzed transcriptomic data from two human bariatric surgery cohorts. In addition, RT-qPCR was performed on adipose tissues from female and male mice fed a high-fat diet. Both visceral and subcutaneous adipose tissue depots were analyzed. Adipose mRNA expression of specific proteoglycans, proteoglycan biosynthetic enzymes, proteoglycan partner molecules, and other ECM-related proteins were altered in both human cohorts. We consistently observed more profound alterations in gene expression of ECM targets in the visceral adipose tissues after surgery (among others VCAN (p = 0.000309), OGN (p = 0.000976), GPC4 (p = 0.00525), COL1A1 (p = 0.00221)). Further, gene analyses in mice revealed sex differences in these two tissue compartments in obese mice. We suggest that adipose tissue repair is still in progress long after surgery, which may reflect challenges in remodeling increased adipose tissues. This study can provide the basis for more mechanistic studies on the role of proteoglycans in adipose tissues in obesity.

Novel insights into adipose tissue heterogeneity.

When normalized to volume, adipose tissue is comprised mainly of large lipid metabolizing and storing cells called adipocytes. Strikingly, the numerical representation of non-adipocytes, composed of a wide variety of cell types found in the so-called stromal vascular fraction (SVF), outnumber adipocytes by far. Besides its function in energy storage, adipose tissue has emerged as a versatile organ that regulates systemic metabolism and has therefore constituted an attractive target for the treatment of metabolic diseases. Recent high-resolution single cells/nucleus RNA seq data exemplify an intriguingly profound diversity of both adipocytes and SVF cells in all adipose depots, and the current data, while limited, demonstrate the significance of the intra-tissue cell composition in shaping the overall functionality of this tissue. Due to the complexity of adipose tissue, our understanding of the biological relevance of this heterogeneity and plasticity is fractional. Therefore, establishing atlases of adipose tissue cell heterogeneity is the first step towards generating an understanding of these functionalities. In this review, we will describe the current knowledge on adipose tissue cell composition and the heterogeneity of single-cell RNA sequencing, including the technical limitations.

Maternal overnutrition programs hedonic and metabolic phenotypes across generations through sperm tsRNAs.

There is a growing body of evidence linking maternal overnutrition to obesity and psychopathology that can be conserved across multiple generations. Recently, we demonstrated in a maternal high-fat diet (HFD; MHFD) mouse model that MHFD induced enhanced hedonic behaviors and obesogenic phenotypes that were conserved across three generations via the paternal lineage, which was independent of sperm methylome changes. Here, we show that sperm tRNA-derived small RNAs (tsRNAs) partly contribute to the transmission of such phenotypes. We observe increased expression of sperm tsRNAs in the F1 male offspring born to HFD-exposed dams. Microinjection of sperm tsRNAs from the F1-HFD male into normal zygotes reproduces obesogenic phenotypes and addictive-like behaviors, such as increased preference of palatable foods and enhanced sensitivity to drugs of abuse in the resultant offspring. The expression of several of the differentially expressed sperm tsRNAs predicted targets such as CHRNA2 and GRIN3A, which have been implicated in addiction pathology, are altered in the mesolimbic reward brain regions of the F1-HFD father and the resultant HFD-tsRNA offspring. Together, our findings demonstrate that sperm tsRNA is a potential vector that contributes to the transmission of MHFD-induced addictive-like behaviors and obesogenic phenotypes across generations, thereby emphasizing its role in diverse pathological outcomes.

Relation of diet-induced thermogenesis to brown adipose tissue activity in healthy men.

Human brown adipose tissue (BAT) is a thermogenic tissue activated by the sympathetic nervous system in response to cold exposure. It contributes to energy expenditure (EE) and takes up glucose and lipids from the circulation. Studies in rodents suggest that BAT contributes to the transient rise in EE after food intake, so-called diet-induced thermogenesis (DIT). We investigated the relationship between human BAT activity and DIT in response to glucose intake in 17 healthy volunteers. We assessed DIT, cold-induced thermogenesis (CIT), and maximum BAT activity at three separate study visits within 2 wk. DIT was measured by indirect calorimetry during an oral glucose tolerance test. CIT was assessed as the difference in EE after cold exposure of 2-h duration as compared with warm conditions. Maximal activity of BAT was assessed by 18-F-fluoro-deoxyglucose (18F-FDG) 18F-FDG-PET/MRI after cold exposure and concomitant pharmacological stimulation with mirabegron. Seventeen healthy men (mean age = 23.4 yr, mean body mass index = 23.2 kg/m2) participated in the study. EE increased from 1,908 (±181) kcal/24 h to 2,128 (±277) kcal/24 h (P < 0.0001, +11.5%) after mild cold exposure. An oral glucose load increased EE from 1,911 (±165) kcal/24 h to 2,096 (±167) kcal/24 h at 60 min (P < 0.0001, +9.7%). The increase in EE in response to cold was significantly associated with BAT activity (R2 = 0.43, P = 0.004). However, DIT was not associated with BAT activity (R2 = 0.015, P = 0.64). DIT after an oral glucose load was not associated with stimulated 18F-FDG uptake into BAT, suggesting that DIT is independent from BAT activity in humans (Clinicaltrials.gov Registration No. NCT03189511).NEW & NOTEWORTHY Cold-induced thermogenesis (CIT) was related to BAT activity as determined by FDG-PET/MRI after stimulation of BAT. Diet-induced thermogenesis (DIT) was not related to stimulated BAT activity. Supraclavicular skin temperature was related to CIT but not to DIT. DIT in humans is probably not a function of BAT.

High-Throughput Single-Cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma.

Even populations of clonal cells are heterogeneous, which requires high-throughput analysis methods with single-cell sensitivity. Here, we propose a rapid, label-free single-cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK-293T, PANC-1, CFPAC-1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.

Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity.

AIMS/HYPOTHESIS: In the context of diabetes, the health benefit of antioxidant treatment has been widely debated. In this study, we investigated the effect of antioxidant treatment during the development of insulin resistance and hyperphagia in obesity and partial lipodystrophy. METHODS: We studied the role of antioxidants in the regulation of insulin resistance using the tamoxifen-inducible fat-specific insulin receptor knockout (iFIRKO) mouse model, which allowed us to analyse the antioxidant's effect in a time-resolved manner. In addition, leptin-deficient ob/ob mice were used as a hyperphagic, chronically obese and diabetic mouse model to validate the beneficial effect of antioxidants on metabolism. RESULTS: Acute induction of insulin receptor knockout in adipocytes changed the substrate preference to fat before induction of a diabetic phenotype including hyperinsulinaemia and hyperglycaemia. In healthy chow-fed animals as well as in morbidly obese mice, this diabetic phase could be reversed within a few weeks. Furthermore, after the induction of insulin receptor knockout in mature adipocytes, iFIRKO mice were protected from subsequent obesity development through high-fat diet feeding. By genetic tracing we show that the persistent fat mass loss in mice after insulin receptor knockout in adipocytes is not caused by the depletion of adipocytes. Treatment of iFIRKO mice with antioxidants postponed and reduced hyperglycaemia by increasing insulin sensitivity. In ob/ob mice, antioxidants rescued both hyperglycaemia and hyperphagia. CONCLUSIONS/INTERPRETATION: We conclude that fat mass reduction through insulin resistance in adipocytes is not reversible. Furthermore, it seems unlikely that adipocytes undergo apoptosis during the process of extreme lipolysis, as a consequence of insulin resistance. Antioxidants have a beneficial health effect not only during the acute phase of diabetes development, but also in a temporary fashion once chronic obesity and diabetes have been established.

Identification of chemotypes in bitter melon by metabolomics: a plant with potential benefit for management of diabetes in traditional Chinese medicine.

INTRODUCTION: Bitter melon (Momordica charantia, Cucurbitaceae) is a popular edible medicinal plant, which has been used as a botanical dietary supplement for the treatment of diabetes and obesity in Chinese folk medicine. Previously, our team has proved that cucurbitanes triterpenoid were involved in bitter melon's anti-diabetic effects as well as on increasing energy expenditure. The triterpenoids composition can however be influenced by changes of varieties or habitats. OBJECTIVES: To clarify the significance of bioactive metabolites diversity among different bitter melons and to provide a guideline for selection of bitter melon varieties, an exploratory study was carried out using a UHPLC-HRMS based metabolomic study to identify chemotypes. METHODS: Metabolites of 55 seed samples of bitter melon collected in different parts of China were profiled by UHPLC-HRMS. The profiling data were analysed with multivariate (MVA) statistical methods. Principle component analysis (PCA) and hierarchical cluster analysis (HCA) were applied for sample differentiation. Marker compounds were identified by comparing spectroscopic data with isolated compounds, and additional triterpenes were putatively identified by propagating annotations through a molecular network (MN) generated from UHPLC-HRMS & MS/MS metabolite profiling. RESULTS: PCA and HCA provided a good discrimination between bitter melon samples from various origins in China. This study revealed for the first time the existence of two chemotypes of bitter melon. Marker compounds of those two chemotypes were identified at different MSI levels. The combined results of MN and MVA demonstrated that the two chemotypes mainly differ in their richness in cucurbitane versus oleanane triterpenoid glycosides (CTGs vs. OTGs). CONCLUSION: Our finding revealed a clear chemotype distribution of bioactive components across bitter melon varieties. While bioactivities of individual CTGs and OTGs still need to be investigated in more depth, our results could help in future the selection of bitter melon varieties with optimised metabolites profile for an improved management of diabetes with this popular edible Chinese folk medicine.