Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of ß cell function.

Circadian rhythm disruption (CD) is associated with impaired glucose homeostasis and type 2 diabetes mellitus (T2DM). While the link between CD and T2DM remains unclear, there is accumulating evidence that disruption of fasting/feeding cycles mediates metabolic dysfunction. Here, we used an approach encompassing analysis of behavioral, physiological, transcriptomic, and epigenomic effects of CD and consequences of restoring fasting/feeding cycles through time-restricted feeding (tRF) in mice. Results show that CD perturbs glucose homeostasis through disruption of pancreatic ß cell function and loss of circadian transcriptional and epigenetic identity. In contrast, restoration of fasting/feeding cycle prevented CD-mediated dysfunction by reestablishing circadian regulation of glucose tolerance, ß cell function, transcriptional profile, and reestablishment of proline and acidic amino acid­rich basic leucine zipper (PAR bZIP) transcription factor DBP expression/activity. This study provides mechanistic insights into circadian regulation of ß cell function and corresponding beneficial effects of tRF in prevention of T2DM.

Electrogenic sodium bicarbonate cotransporter NBCe1 regulates pancreatic ß cell function in type 2 diabetes.

Pancreatic ß cell failure in type 2 diabetes mellitus (T2DM) is attributed to perturbations of the ß cell's transcriptional landscape resulting in impaired glucose-stimulated insulin secretion. Recent studies identified SLC4A4 (a gene encoding an electrogenic Na+-coupled HCO3- cotransporter and intracellular pH regulator, NBCe1) as one of the misexpressed genes in ß cells of patients with T2DM. Thus, in the current study, we set out to test the hypothesis that misexpression of SLC4A4/NBCe1 in T2DM ß cells contributes to ß cell dysfunction and impaired glucose homeostasis. To address this hypothesis, we first confirmed induction of SLC4A4/NBCe1 expression in ß cells of patients with T2DM and demonstrated that its expression was associated with loss of ß cell transcriptional identity, intracellular alkalinization, and ß cell dysfunction. In addition, we generated a ß cell-selective Slc4a4/NBCe1-KO mouse model and found that these mice were protected from diet-induced metabolic stress and ß cell dysfunction. Importantly, improved glucose tolerance and enhanced ß cell function in Slc4a4/NBCe1-deficient mice were due to augmented mitochondrial function and increased expression of genes regulating ß cell identity and function. These results suggest that increased ß cell expression of SLC4A4/NBCe1 in T2DM plays a contributory role in promotion of ß cell failure and should be considered as a potential therapeutic target.

Pro-inflammatory ß cell small extracellular vesicles induce ß cell failure through activation of the CXCL10/CXCR3 axis in diabetes.

Coordinated communication among pancreatic islet cells is necessary for maintenance of glucose homeostasis. In diabetes, chronic exposure to pro-inflammatory cytokines has been shown to perturb ß cell communication and function. Compelling evidence has implicated extracellular vesicles (EVs) in modulating physiological and pathological responses to ß cell stress. We report that pro-inflammatory ß cell small EVs (cytokine-exposed EVs [cytoEVs]) induce ß cell dysfunction, promote a pro-inflammatory islet transcriptome, and enhance recruitment of CD8+ T cells and macrophages. Proteomic analysis of cytoEVs shows enrichment of the chemokine CXCL10, with surface topological analysis depicting CXCL10 as membrane bound on cytoEVs to facilitate direct binding to CXCR3 receptors on the surface of ß cells. CXCR3 receptor inhibition reduced CXCL10-cytoEV binding and attenuated ß cell dysfunction, inflammatory gene expression, and leukocyte recruitment to islets. This work implies a significant role of pro-inflammatory ß cell-derived small EVs in modulating ß cell function, global gene expression, and antigen presentation through activation of the CXCL10/CXCR3 axis.

Proinflammatory Cytokine Interleukin 1ß Disrupts ß-cell Circadian Clock Function and Regulation of Insulin Secretion.

Intrinsic ß-cell circadian clocks are important regulators of insulin secretion and overall glucose homeostasis. Whether the circadian clock in ß-cells is perturbed following exposure to prodiabetogenic stressors such as proinflammatory cytokines, and whether these perturbations are featured during the development of diabetes, remains unknown. To address this, we examined the effects of cytokine-mediated inflammation common to the pathophysiology of diabetes, on the physiological and molecular regulation of the ß-cell circadian clock. Specifically, we provide evidence that the key diabetogenic cytokine IL-1ß disrupts functionality of the ß-cell circadian clock and impairs circadian regulation of glucose-stimulated insulin secretion. The deleterious effects of IL-1ß on the circadian clock were attributed to impaired expression of key circadian transcription factor Bmal1, and its regulator, the NAD-dependent deacetylase, Sirtuin 1 (SIRT1). Moreover, we also identified that Type 2 diabetes in humans is associated with reduced immunoreactivity of ß-cell BMAL1 and SIRT1, suggestive of a potential causative link between islet inflammation, circadian clock disruption, and ß-cell failure. These data suggest that the circadian clock in ß-cells is perturbed following exposure to proinflammatory stressors and highlights the potential for therapeutic targeting of the circadian system for treatment for ß-cell failure in diabetes.

Publisher Correction: Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phases of Metabolic and Soft Tissue Changes in Months Preceding a Diagnosis of Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Identifying metabolic abnormalities that occur before pancreatic ductal adenocarcinoma (PDAC) diagnosis could increase chances for early detection. We collected data on changes in metabolic parameters (glucose, serum lipids, triglycerides; total, low-density, and high-density cholesterol; and total body weight) and soft tissues (abdominal subcutaneous fat [SAT], adipose tissue, visceral adipose tissue [VAT], and muscle) from patients 5 years before the received a diagnosis of PDAC. METHODS: We collected data from 219 patients with a diagnosis of PDAC (patients) and 657 healthy individuals (controls) from the Rochester Epidemiology Project, from 2000 through 2015. We compared metabolic profiles of patients with those of age- and sex-matched controls, constructing temporal profiles of fasting blood glucose, serum lipids including triglycerides, cholesterol profiles, and body weight and temperature for 60 months before the diagnosis of PDAC (index date). To construct the temporal profile of soft tissue changes, we collected computed tomography scans from 68 patients, comparing baseline (>18 months before diagnosis) areas of SAT, VAT, and muscle at L2/L3 vertebra with those of later scans until time of diagnosis. SAT and VAT, isolated from healthy individuals, were exposed to exosomes isolated from PDAC cell lines and analyzed by RNA sequencing. SAT was collected from KRAS+/LSLG12D P53flox/flox mice with PDACs, C57/BL6 (control) mice, and 5 patients and analyzed by histology and immunohistochemistry. RESULTS: There were no significant differences in metabolic or soft tissue features of patients vs controls until 30 months before PDAC diagnosis. In the 30 to 18 months before PDAC diagnosis (phase 1, hyperglycemia), a significant proportion of patients developed hyperglycemia, compared with controls, without soft tissue changes. In the 18 to 6 months before PDAC diagnosis (phase 2, pre-cachexia), patients had significant increases in hyperglycemia and decreases in serum lipids, body weight, and SAT, with preserved VAT and muscle. In the 6 to 0 months before PDAC diagnosis (phase 3, cachexia), a significant proportion of patients had hyperglycemia compared with controls, and patients had significant reductions in all serum lipids, SAT, VAT, and muscle. We believe the patients had browning of SAT, based on increases in body temperature, starting 18 months before PDAC diagnosis. We observed expression of uncoupling protein 1 (UCP1) in SAT exposed to PDAC exosomes, SAT from mice with PDACs, and SAT from all 5 patients but only 1 of 4 controls. CONCLUSIONS: We identified 3 phases of metabolic and soft tissue changes that precede a diagnosis of PDAC. Loss of SAT starts 18 months before PDAC identification, and is likely due to browning. Overexpression of UCP1 in SAT might be a biomarker of early-stage PDAC, but further studies are needed.

Shedding Perspective on Extracellular Vesicle Biology in Diabetes and Associated Metabolic Syndromes.

The etiology of diabetes and associated metabolic derailments is a complex process that relies on crosstalk between metabolically active tissues. Dysregulation of secreted factors and metabolites from islets, adipose tissue, liver, and skeletal muscle contributes to the overall progression of diabetes and metabolic syndrome. Extracellular vesicles (EVs) are circulating nanovesicles secreted by most cell types and are comprised of bioactive cargoes that are horizontally transferred to targeted cells/tissues. Accumulating evidence from the past decade implicates the role of EVs as mediators of islet cell dysfunction, inflammation, insulin resistance, and other metabolic consequences associated with diabetes. This review covers a broad spectrum of basic EV biology (i.e., biogenesis, secretion, and uptake), including a comprehensive investigation of the emerging role of EVs in ß-cell autocrine/paracrine interactions and the multidirectional crosstalk in metabolically active tissues. Understanding the utility of this novel means of intercellular communication could impart insight into the development of new treatment regimens and biomarker detection to treat diabetes.

Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

ß-cell proliferation induction is a promising therapeutic strategy to restore ß-cell mass. By screening small molecules in a transgenic zebrafish model of type 1 diabetes, we identified inhibitors of non-canonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), as enhancers of ß-cell regeneration. The most potent ß-cell regeneration enhancer was a cinnamic acid derivative (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA), which, acting through the cAMP-dependent protein kinase A (PKA), stimulated ß-cell-specific proliferation by increasing cyclic AMP (cAMP) levels and mechanistic target of rapamycin (mTOR) activity. A combination of PIAA and cilostamide, an inhibitor of ß-cell-enriched cAMP hydrolyzing enzyme phosphodiesterase (PDE) 3, enhanced ß-cell proliferation, whereas overexpression of PDE3 blunted the mitogenic effect of PIAA in zebrafish. PIAA augmented proliferation of INS-1ß-cells and ß-cells in mammalian islets including human islets with elevation in cAMP levels and insulin secretion. PIAA improved glycemic control in streptozotocin (STZ)-induced diabetic mice with increases in ß-cell proliferation, ß-cell area, and insulin content in the pancreas. Collectively, these data reveal an evolutionarily conserved and critical role of TBK1/IKKε suppression in expanding functional ß-cell mass.

Circadian Etiology of Type 2 Diabetes Mellitus.

The epidemic of Type 2 diabetes mellitus necessitates development of novel therapeutic and preventative strategies to attenuate expansion of this debilitating disease. Evidence links the circadian system to various aspects of diabetes pathophysiology and treatment. The aim of this review will be to outline the rationale for therapeutic targeting of the circadian system in the treatment and prevention of Type 2 diabetes mellitus and consequent metabolic comorbidities.

Exosomes and their role in the micro-/macro-environment: a comprehensive review.

The importance of extracellular vesicles (EVs) in cell-cell communication has long been recognized due to their ability to transfer important cellular cargoes such as DNA, mRNA, miRNAs, and proteins to target cells. Compelling evidence supports the role of EVs in the horizontal transfer of cellular material which has the potential to influence normal cellular physiology and promote various disease states. Of the different types of EVs, exosomes have garnered much attention in the past decade due to their abundance in various biological fluids and ability to affect multiple organ systems. The main focus of this review will be on cancer and how cancer-derived exosomes are important mediators of metastasis, angiogenesis, immune modulation, and the tumor macro-/microenvironment. We will also discuss exosomes as potential biomarkers for cancers due to their abundance in biological fluids, ease of uptake, and cellular content. Exosome use in diagnosis, prognosis, and in establishing treatment regimens has enormous potential to revolutionize patient care.

Immunosuppressive CD14+HLA-DRlo/neg monocytes are elevated in pancreatic cancer and "primed" by tumor-derived exosomes.

Immunological strategies to treat pancreatic cancer offer new therapeutic approaches to improve patient outcomes. Understanding alterations in the immune systems of pancreatic cancer patients will likely lead to advances in immunotherapy for the disease. We profiled peripheral blood leukocytes from pancreatic cancer patients (n = 22) and age-matched controls (n = 20) using flow cytometry. Immune profiling of pancreatic cancer patients identified phenotypic changes in various immune cell populations, including a population of immunosuppressive monocytes (CD14+HLA-DRlo/neg), which were shown to be increased in these patients. There was a correlation between the levels of CD14+ monocytes and the levels of CD14+HLA-DRlo/neg monocytes in peripheral blood from pancreatic cancer patients. HLA-DR downregulation of monocytes was shown to occur through pancreatic cancer-derived exosome interactions with monocytes. In an in vitro model, exosomes from patient-derived xenograft cell lines and patient plasma decreased HLA-DR expression on CD14+ monocytes. Additionally, tumor-derived exosomes caused immune suppression in monocytes through altered STAT3 signaling, induction of arginase expression, and reactive oxygen species. These findings provide novel insights into the mechanisms that govern immunosuppression in pancreatic cancer. Understanding monocyte-exosome interactions could lead to novel immunotherapies for this disease.

Pathogenesis of pancreatic cancer exosome-induced lipolysis in adipose tissue.

BACKGROUND AND OBJECTIVES: New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. DESIGN: Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. RESULTS: In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. CONCLUSIONS: PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.

Controlled expression of Drosophila homeobox loci using the Hostile takeover system.

BACKGROUND: Hostile takeover (Hto) is a Drosophila protein trapping system that allows the investigator to both induce a gene and tag its product. The Hto transposon carries a GAL4-regulated promoter expressing an exon encoding a FLAG-mCherry tag. Upon expression, the Hto exon can splice to a downstream genomic exon, generating a fusion transcript and tagged protein. RESULTS: Using rough-eye phenotypic screens, Hto inserts were recovered at eight homeobox or Pax loci: cut, Drgx/CG34340, Pox neuro, araucan, shaven/D-Pax2, Zn finger homeodomain 2, Sex combs reduced (Scr), and the abdominal-A region. The collection yields diverse misexpression phenotypes. Ectopic Drgx was found to alter the cytoskeleton and cell adhesion in ovary follicle cells. Hto expression of cut, araucan, or shaven gives phenotypes similar to those of the corresponding UAS-cDNA constructs. The cut and Pox neuro phenotypes are suppressed by the corresponding RNAi constructs. The Scr and abdominal-A inserts do not make fusion proteins, but may act by chromatin- or RNA-based mechanisms. CONCLUSIONS: Hto can effectively express tagged homeodomain proteins from their endogenous loci; the Minos vector allows inserts to be obtained even in transposon cold-spots. Hto screens may recover homeobox genes at high rates because they are particularly sensitive to misexpression.

Dopamine D2 receptor agonists inhibit lung cancer progression by reducing angiogenesis and tumor infiltrating myeloid derived suppressor cells.

We sought to determine whether Dopamine D2 Receptor (D2R) agonists inhibit lung tumor progression and identify subpopulations of lung cancer patients that benefit most from D2R agonist therapy. We demonstrate D2R agonists abrogate lung tumor progression in syngeneic (LLC1) and human xenograft (A549) orthotopic murine models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells. Pathological examination of human lung cancer tissue revealed a positive correlation between endothelial D2R expression and tumor stage. Lung cancer patients with a smoking history exhibited greater levels of D2R in lung endothelium. Our results suggest D2R agonists may represent a promising individualized therapy for lung cancer patients with high levels of endothelial D2R expression and a smoking history.

Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic ß-cell Dysfunction.

PURPOSE: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic ß-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches ß cells remote from the cancer to induce ß-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to ß cells and impair insulin secretion. EXPERIMENTAL METHODS: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 ß cells and human islets, and demonstrated the mechanism of exosome internalization into ß cells. We studied the interaction between ß-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in ß cells was shown. RESULTS: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered ß cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on ß cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. ß cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. CONCLUSIONS: Pancreatic cancer causes paraneoplastic ß-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response.

Inducible protein traps with dominant phenotypes for functional analysis of the Drosophila genome.

The Drosophila melanogaster genome has been extensively characterized, but there remains a pressing need to associate gene products with phenotypes, subcellular localizations, and interaction partners. A multifunctional, Minos transposon-based protein trapping system called Hostile takeover (Hto) was developed to facilitate in vivo analyses of endogenous genes, including live imaging, purification of protein complexes, and mutagenesis. The Hto transposon features a UAS enhancer with a basal promoter, followed by an artificial exon 1 and a standard 5' splice site. Upon GAL4 induction, exon 1 can splice to the next exon downstream in the flanking genomic DNA, belonging to a random target gene. Exon 1 encodes a dual tag (FLAG epitope and mCherry red fluorescent protein), which becomes fused to the target protein. Hto was mobilized throughout the genome and then activated by eye-specific GAL4; an F1 screen for abnormal eye phenotypes was used to identify inserts that express disruptive fusion proteins. Approximately 1.7% of new inserts cause eye phenotypes. Of the first 23 verified target genes, 21 can be described as regulators of cell biology and development. Most are transcription factor genes, including AP-2, CG17181, cut, klu, mamo, Sox102F, and sv. Other target genes [l(1)G0232, nuf, pum, and Syt4] make cytoplasmic proteins, and these lines produce diverse fluorescence localization patterns. Hto permits the expression of stable carboxy-terminal subfragments of proteins, which are rarely tested in conventional genetic screens. Some of these may disrupt specific cell pathways, as exemplified by truncated forms of Mastermind and Nuf.

Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in ß cells and mice.

BACKGROUND & AIMS: New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer. METHODS: Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls). RESULTS: Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not. CONCLUSIONS: Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in ß cells and mice.