Age-associated insulin resistance (IR) and obesity-associated IR are two physiologically distinct forms of adult-onset diabetes. While macrophage-driven inflammation is a core driver of obesity-associated IR, the underlying mechanisms of the obesity-independent yet highly prevalent age-associated IR are largely unexplored. Here we show, using comparative adipo-immune profiling in mice, that fat-resident regulatory T cells, termed fTreg cells, accumulate in adipose tissue as a function of age, but not obesity. Supporting the existence of two distinct mechanisms underlying IR, mice deficient in fTreg cells are protected against age-associated IR, yet remain susceptible to obesity-associated IR and metabolic disease. By contrast, selective depletion of fTreg cells via anti-ST2 antibody treatment increases adipose tissue insulin sensitivity. These findings establish that distinct immune cell populations within adipose tissue underlie ageing- and obesity-associated IR, and implicate fTreg cells as adipo-immune drivers and potential therapeutic targets in the treatment of age-associated IR.

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Islets derived from stem cells hold promise as a therapy for insulin-dependent diabetes, but there remain challenges towards achieving this goal1-6. Here we generate human islet-like organoids (HILOs) from induced pluripotent stem cells and show that non-canonical WNT4 signalling drives the metabolic maturation necessary for robust ex vivo glucose-stimulated insulin secretion. These functionally mature HILOs contain endocrine-like cell types that, upon transplantation, rapidly re-establish glucose homeostasis in diabetic NOD/SCID mice. Overexpression of the immune checkpoint protein programmed death-ligand 1 (PD-L1) protected HILO xenografts such that they were able to restore glucose homeostasis in immune-competent diabetic mice for 50 days. Furthermore, ex vivo stimulation with interferon-γ induced endogenous PD-L1 expression and restricted T cell activation and graft rejection. The generation of glucose-responsive islet-like organoids that are able to avoid immune detection provides a promising alternative to cadaveric and device-dependent therapies in the treatment of diabetes.

Fibroblast growth factor 1 (FGF1) is an autocrine/paracrine regulator whose binding to heparan sulphate proteoglycans effectively precludes its circulation. Although FGF1 is known as a mitogenic factor, FGF1 knockout mice develop insulin resistance when stressed by a high-fat diet, suggesting a potential role in nutrient homeostasis. Here we show that parenteral delivery of a single dose of recombinant FGF1 (rFGF1) results in potent, insulin-dependent lowering of glucose levels in diabetic mice that is dose-dependent but does not lead to hypoglycaemia. Chronic pharmacological treatment with rFGF1 increases insulin-dependent glucose uptake in skeletal muscle and suppresses the hepatic production of glucose to achieve whole-body insulin sensitization. The sustained glucose lowering and insulin sensitization attributed to rFGF1 are not accompanied by the side effects of weight gain, liver steatosis and bone loss associated with current insulin-sensitizing therapies. We also show that the glucose-lowering activity of FGF1 can be dissociated from its mitogenic activity and is mediated predominantly via FGF receptor 1 signalling. Thus we have uncovered an unexpected, neomorphic insulin-sensitizing action for exogenous non-mitogenic human FGF1 with therapeutic potential for the treatment of insulin resistance and type 2 diabetes.

Mammalian metabolism is highly circadian and major hormonal circuits involving nuclear hormone receptors display interlinked diurnal cycling. However, mechanisms that logically explain the coordination of nuclear hormone receptors and the clock are poorly understood. Here we show that two circadian co-regulators, cryptochromes 1 and 2, interact with the glucocorticoid receptor in a ligand-dependent fashion and globally alter the transcriptional response to glucocorticoids in mouse embryonic fibroblasts: cryptochrome deficiency vastly decreases gene repression and approximately doubles the number of dexamethasone-induced genes, suggesting that cryptochromes broadly oppose glucocorticoid receptor activation and promote repression. In mice, genetic loss of cryptochrome 1 and/or 2 results in glucose intolerance and constitutively high levels of circulating corticosterone, suggesting reduced suppression of the hypothalamic-pituitary-adrenal axis coupled with increased glucocorticoid transactivation in the liver. Genomically, cryptochromes 1 and 2 associate with a glucocorticoid response element in the phosphoenolpyruvate carboxykinase 1 promoter in a hormone-dependent manner, and dexamethasone-induced transcription of the phosphoenolpyruvate carboxykinase 1 gene was strikingly increased in cryptochrome-deficient livers. These results reveal a specific mechanism through which cryptochromes couple the activity of clock and receptor target genes to complex genomic circuits underpinning normal metabolic homeostasis.

The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK-BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-α and REV-ERB-β have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with the master circadian regulator BMAL1. Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-β genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-β cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-β function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-α and REV-ERB-β with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.

Although feast and famine cycles illustrate that remodelling of adipose tissue in response to fluctuations in nutrient availability is essential for maintaining metabolic homeostasis, the underlying mechanisms remain poorly understood. Here we identify fibroblast growth factor 1 (FGF1) as a critical transducer in this process in mice, and link its regulation to the nuclear receptor PPARγ (peroxisome proliferator activated receptor γ), which is the adipocyte master regulator and the target of the thiazolidinedione class of insulin sensitizing drugs. FGF1 is the prototype of the 22-member FGF family of proteins and has been implicated in a range of physiological processes, including development, wound healing and cardiovascular changes. Surprisingly, FGF1 knockout mice display no significant phenotype under standard laboratory conditions. We show that FGF1 is highly induced in adipose tissue in response to a high-fat diet and that mice lacking FGF1 develop an aggressive diabetic phenotype coupled to aberrant adipose expansion when challenged with a high-fat diet. Further analysis of adipose depots in FGF1-deficient mice revealed multiple histopathologies in the vasculature network, an accentuated inflammatory response, aberrant adipocyte size distribution and ectopic expression of pancreatic lipases. On withdrawal of the high-fat diet, this inflamed adipose tissue fails to properly resolve, resulting in extensive fat necrosis. In terms of mechanisms, we show that adipose induction of FGF1 in the fed state is regulated by PPARγ acting through an evolutionarily conserved promoter proximal PPAR response element within the FGF1 gene. The discovery of a phenotype for the FGF1 knockout mouse establishes the PPARγ–FGF1 axis as critical for maintaining metabolic homeostasis and insulin sensitization.

Adipocytes increase energy expenditure in response to prolonged sympathetic activation via persistent expression of uncoupling protein 1 (UCP1)1,2. Here we report that the regulation of glycogen metabolism by catecholamines is critical for UCP1 expression. Chronic β-adrenergic activation leads to increased glycogen accumulation in adipocytes expressing UCP1. Adipocyte-specific deletion of a scaffolding protein, protein targeting to glycogen (PTG), reduces glycogen levels in beige adipocytes, attenuating UCP1 expression and responsiveness to cold or β-adrenergic receptor-stimulated weight loss in obese mice. Unexpectedly, we observed that glycogen synthesis and degradation are increased in response to catecholamines, and that glycogen turnover is required to produce reactive oxygen species leading to the activation of p38 MAPK, which drives UCP1 expression. Thus, glycogen has a key regulatory role in adipocytes, linking glucose metabolism to thermogenesis.