Single-cell transcriptome analysis reveals secretin as a hallmark of human enteroendocrine cell maturation.

The traditional nomenclature of enteroendocrine cells (EECs), established in 1977, applied the "one cell - one hormone" dogma, which distinguishes subpopulations based on the secretion of a specific hormone. These hormone-specific subpopulations included S cells for secretin (SCT), K cells for glucose-dependent insulinotropic polypeptide (GIP), N cells producing neurotensin (NTS), I cells producing cholecystokinin (CCK), D cells producing somatostatin (SST), and others. In the past 15 years, reinvestigations into murine and human organoid-derived EECs, however, strongly questioned this dogma and established that certain EECs coexpress multiple hormones. Using the Gut Cell Atlas, the largest available single-cell transcriptome dataset of human intestinal cells, this study consolidates that the original dogma is outdated not only for murine and human organoid-derived EECs, but also for primary human EECs, showing that the expression of certain hormones is not restricted to their designated cell type. Moreover, specific analyses into SCT-expressing cells reject the presence of any cell population that exhibits significantly elevated secretin expression compared to other cell populations, previously referred to as S cells. Instead, this investigation indicates that secretin production is realized jointly by other enteroendocrine subpopulations, validating corresponding observations in murine EECs also for human EECs. Furthermore, our findings corroborate that SCT expression peaks in mature EECs, in contrast, progenitor EECs exhibit markedly lower expression levels, supporting the hypothesis that SCT expression is a hallmark of EEC maturation.

LncRNA Ctcflos orchestrates transcription and alternative splicing in thermogenic adipogenesis.

The recruitment of thermogenic brite adipocytes within white adipose tissue attenuates obesity and metabolic comorbidities, arousing interest in understanding the underlying regulatory mechanisms. The molecular network of brite adipogenesis, however, remains largely unresolved. In this light, long noncoding RNAs (lncRNAs) emerged as a versatile class of modulators that control many steps within the differentiation machinery. Leveraging the naturally varying propensities of different inbred mouse strains for white adipose tissue browning, we identify the nuclear lncRNA Ctcflos as a pivotal orchestrator of thermogenic gene expression during brite adipocyte differentiation. Mechanistically, Ctcflos acts as a pleiotropic regulator, being essential for the transcriptional recruitment of the early core thermogenic regulatory program and the modulation of alternative splicing to drive brite adipogenesis. This is showcased by Ctcflos-regulated gene transcription and splicing of the key browning factor Prdm16 toward the isoform that is specific for the thermogenic gene program. Conclusively, our findings emphasize the mechanistic versatility of lncRNAs acting at several independent levels of gene expression for effective regulation of key differentiation factors to direct cell fate and function.

Uncoupling protein-1 expression does not protect mice from diet-induced obesity.

We studied the metabolic phenotype of a novel Ucp1-LUC-iRFP713 knock-in reporter gene mouse model originally generated to monitor endogenous Ucp1 gene expression. Both reporter mice and reporter cells reliably reflected Ucp1 gene expression in vivo and in vitro. We here report an unexpected reduction in UCP1 content in homozygous knock-in (KI) reporter mice. As a result, the thermogenic capacity of KI mice stimulated by norepinephrine was largely blunted, making them more sensitive to an acute cold exposure. In return, these reporter mice with reduced UCP1 expression enabled us to investigate the physiological role of UCP1 in the prevention of weight gain. We observed no substantial differences in body mass across the three genotypes, irrespective of the type of diet or the ambient temperature, possibly due to the insufficient UCP1 activation. Indeed, activation of UCP1 by daily injection of the selective ß3-adrenergic receptor agonist CL316,243 resulted in significantly greater reduction of body weight in wild-type mice than in KI mice. Taken together, we conclude that the intact expression of UCP1 is essential for cold-induced thermogenesis but the presence of UCP1 per se does not protect mice from diet-induced obesity.NEW & NOTEWORTHY To study the functional role of UCP1-dependent brown adipose tissue thermogenesis for energy balance, new animal models are needed. By metabolic phenotyping of a novel mouse model with low UCP1 levels in brown fat, we demonstrate that the susceptibility to diet-induced obesity is not increased despite impaired cold-induced thermogenic capacity. Brown fat requires pharmacological activation to promote negative energy balance in diet-induced obese mice.

Isolation, Culture, and Functional Analysis of Murine Thermogenic Adipocytes.

Studying brown and brite adipose tissue requires precise and reliable quantification of cellular thermogenesis. This protocol describes the isolation of primary murine pre-adipocytes, differentiation into thermogenic brown and brite adipocytes, and subsequent oxygen consumption analysis. Commonly applied procedures only measure basal and maximal proton leak-linked oxygen consumption but not explicitly uncoupling protein 1 (UCP1)-dependent respiration. Meaningful oxygen consumption analyses require (1) the activation of UCP1, (2) control over intracellular free-fatty-acid levels, and (3) inhibition of ATP-consuming futile cycles. For complete details on the use and execution of this protocol, please refer to Li et al. (2014, 2017, 2018) and Schweizer et al. (2018).

Purine Nucleotides in the Regulation of Brown Adipose Tissue Activity.

Non-shivering thermogenesis in mammalian brown adipose tissue is a powerful mechanism to defend normothermia in cold climates. To minimize the loss of chemical energy, the central functional component, mitochondrial uncoupling protein 1, UCP1, must be tightly regulated. The canonical pathway of UCP1 activation includes lipolytic release of free fatty acids in response to an adrenergic signal. Activating fatty acids overcome constitutive inhibition of UCP1 by the di- and triphosphate forms of purine nucleotides, i.e., ATP, ADP, GTP, and GDP. Cellular concentrations of inhibitory, free nucleotides are subject to significant, adrenergically induced alterations. The regulatory components involved may constitute novel drug targets to manipulate brown fat thermogenesis and thereby organismic energy balance. We here review evidence for and against a dominant role of nucleotides in thermogenic control, describe conceptual routes to endogenously and pharmacologically alter free nucleotide pool size, speculate on a signaling role of degradation products released from active brown fat, and highlight gaps in our understanding of signaling and metabolic pathways involved.

Systems-Genetics-Based Inference of a Core Regulatory Network Underlying White Fat Browning.

Recruitment of brite/beige cells, known as browning of white adipose tissue (WAT), is an efficient way to turn an energy-storing organ into an energy-dissipating one and may therefore be of therapeutic value in combating obesity. However, a comprehensive understanding of the regulatory mechanisms mediating WAT browning is still lacking. Here, we exploit the large natural variation in WAT browning propensity between inbred mouse strains to gain an inclusive view of the core regulatory network coordinating this cellular process. Combining comparative transcriptomics, perturbation-based validations, and gene network analyses, we present a comprehensive gene regulatory network of inguinal WAT browning, revealing up to four distinct regulatory modules with key roles for uncovered transcriptional factors, while also providing deep insights into the genetic architecture of brite adipogenesis. The presented findings therefore greatly increase our understanding of the molecular drivers mediating the intriguing cellular heterogeneity and plasticity of adipose tissue.

Secretin-Activated Brown Fat Mediates Prandial Thermogenesis to Induce Satiation.

The molecular mediator and functional significance of meal-associated brown fat (BAT) thermogenesis remains elusive. Here, we identified the gut hormone secretin as a non-sympathetic BAT activator mediating prandial thermogenesis, which consequentially induces satiation, thereby establishing a gut-secretin-BAT-brain axis in mammals with a physiological role of prandial thermogenesis in the control of satiation. Mechanistically, meal-associated rise in circulating secretin activates BAT thermogenesis by stimulating lipolysis upon binding to secretin receptors in brown adipocytes, which is sensed in the brain and promotes satiation. Chronic infusion of a modified human secretin transiently elevates energy expenditure in diet-induced obese mice. Clinical trials with human subjects showed that thermogenesis after a single-meal ingestion correlated with postprandial secretin levels and that secretin infusions increased glucose uptake in BAT. Collectively, our findings highlight the largely unappreciated function of BAT in the control of satiation and qualify BAT as an even more attractive target for treating obesity.