Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

Lysophosphatidylinositols Are Upregulated After Human ß-Cell Loss and Potentiate Insulin Release.

In this study, we identified new lipid species associated with the loss of pancreatic ß-cells triggering diabetes. We performed lipidomics measurements on serum from prediabetic mice lacking ß-cell prohibitin-2 (a model of monogenic diabetes) patients without previous history of diabetes but scheduled for pancreaticoduodenectomy resulting in the acute reduction of their ß-cell mass (∼50%), and patients with type 2 diabetes (T2D). We found lysophosphatidylinositols (lysoPIs) were the main circulating lipid species altered in prediabetic mice. The changes were confirmed in the patients with acute reduction of their ß-cell mass and in those with T2D. Increased lysoPIs significantly correlated with HbA1c (reflecting glycemic control), fasting glycemia, and disposition index, and did not correlate with insulin resistance or obesity in human patients with T2D. INS-1E ß-cells as well as pancreatic islets isolated from nondiabetic mice and human donors exposed to exogenous lysoPIs showed potentiated glucose-stimulated and basal insulin secretion. Finally, addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. Overall, lysoPIs appear to be lipid species upregulated in the prediabetic stage associated with the loss of ß-cells and that support the secretory function of the remaining ß-cells. ARTICLE HIGHLIGHTS: Circulating lysophosphatidylinositols (lysoPIs) are increased in situations associated with ß-cell loss in mice and humans such as (pre-)diabetes, and hemipancreatectomy. Pancreatic islets isolated from nondiabetic mice and human donors, as well as INS-1E ß-cells, exposed to exogenous lysoPIs exhibited potentiated glucose-stimulated and basal insulin secretion. Addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. LysoPIs appear as lipid species being upregulated already in the prediabetic stage associated with the loss of ß-cells and supporting the function of the remaining ß-cells.

Circadian organization of lipid landscape is perturbed in type 2 diabetic patients.

Lipid homeostasis in humans follows a diurnal pattern in muscle and pancreatic islets, altered upon metabolic dysregulation. We employ tandem and liquid-chromatography mass spectrometry to investigate daily regulation of lipid metabolism in subcutaneous white adipose tissue (SAT) and serum of type 2 diabetic (T2D) and non-diabetic (ND) human volunteers (n = 12). Around 8% of ≈440 lipid metabolites exhibit diurnal rhythmicity in serum and SAT from ND and T2D subjects. The spectrum of rhythmic lipids differs between ND and T2D individuals, with the most substantial changes observed early morning, as confirmed by lipidomics in an independent cohort of ND and T2D subjects (n = 32) conducted at a single morning time point. Strikingly, metabolites identified as daily rhythmic in both serum and SAT from T2D subjects exhibit phase differences. Our study reveals massive temporal and tissue-specific alterations of human lipid homeostasis in T2D, providing essential clues for the development of lipid biomarkers in a temporal manner.

Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis.

Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient-sensing signalling pathways, yet mechanisms of their interaction remain not fully understood. Surprisingly, we find that class 3 phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in gene transcription as a coactivator of the heterodimeric transcription factor and circadian driver Bmal1-Clock. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between the lipid kinase Vps34 and regulatory subunit Vps15. We demonstrate that although both subunits of class 3 PI3K interact with RNA polymerase II and co-localize with active transcription sites, exclusive loss of Vps15 in cells blunts the transcriptional activity of Bmal1-Clock. Thus, we establish non-redundancy between nuclear Vps34 and Vps15, reflected by the persistent nuclear pool of Vps15 in Vps34-depleted cells and the ability of Vps15 to coactivate Bmal1-Clock independently of its complex with Vps34. In physiology we find that Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promotes pro-anabolic de novo purine nucleotide synthesis. We show that Vps15 activates the transcription of Ppat, a key enzyme for the production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, Vps15 levels are decreased on the promoters of Bmal1 targets, Nr1d1 and Ppat. Our findings open avenues for establishing the complexity for nuclear class 3 PI3K signalling for temporal regulation of energy homeostasis.

Lipid metabolism around the body clocks.

Lipids play important roles in energy metabolism along with diverse aspects of biological membrane structure, signaling and other functions. Perturbations of lipid metabolism are responsible for the development of various pathologies comprising metabolic syndrome, obesity, and type 2 diabetes. Accumulating evidence suggests that circadian oscillators, operative in most cells of our body, coordinate temporal aspects of lipid homeostasis. In this review we summarize current knowledge on the circadian regulation of lipid digestion, absorption, transportation, biosynthesis, catabolism, and storage. Specifically, we focus on the molecular interactions between functional clockwork and biosynthetic pathways of major lipid classes comprising cholesterol, fatty acids, triacylglycerols, glycerophospholipids, glycosphingolipids, and sphingomyelins. A growing body of epidemiological studies associate a socially imposed circadian misalignment common in modern society with growing incidence of metabolic disorders, however the disruption of lipid metabolism rhythms in this connection has only been recently revealed. Here, we highlight recent studies that unravel the mechanistic link between intracellular molecular clocks, lipid homeostasis and development of metabolic diseases based on animal models of clock disruption and on innovative translational studies in humans. We also discuss the perspectives of manipulating circadian oscillators as a potentially powerful approach for preventing and managing metabolic disorders in human patients.

Influence of circadian clocks on adaptive immunity and vaccination responses.

The adaptive immune response is under circadian control, yet, why adaptive immune reactions continue to exhibit circadian changes over long periods of time is unknown. Using a combination of experimental and mathematical modeling approaches, we show here that dendritic cells migrate from the skin to the draining lymph node in a time-of-day-dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Rhythmic expression of TNF in the draining lymph node enhances BMAL1-controlled ICAM-1 expression in high endothelial venules, resulting in lymphocyte infiltration and lymph node expansion. Lymph node cellularity continues to be different for weeks after the initial time-of-day-dependent challenge, which governs the immune response to vaccinations directed against Hepatitis A virus as well as SARS-CoV-2. In this work, we present a mechanistic understanding of the time-of-day dependent development and maintenance of an adaptive immune response, providing a strategy for using time-of-day to optimize vaccination regimes.

Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets.

Recent evidence suggests that circadian clocks ensure temporal orchestration of lipid homeostasis and play a role in pathophysiology of metabolic diseases in humans, including type 2 diabetes (T2D). Nevertheless, circadian regulation of lipid metabolism in human pancreatic islets has not been explored. Employing lipidomic analyses, we conducted temporal profiling in human pancreatic islets derived from 10 nondiabetic (ND) and 6 T2D donors. Among 329 detected lipid species across 8 major lipid classes, 5% exhibited circadian rhythmicity in ND human islets synchronized in vitro. Two-time point-based lipidomic analyses in T2D human islets revealed global and temporal alterations in phospho- and sphingolipids. Key enzymes regulating turnover of sphingolipids were rhythmically expressed in ND islets and exhibited altered levels in ND islets bearing disrupted clocks and in T2D islets. Strikingly, cellular membrane fluidity, measured by a Nile Red derivative NR12S, was reduced in plasma membrane of T2D diabetic human islets, in ND donors' islets with disrupted circadian clockwork, or treated with sphingolipid pathway modulators. Moreover, inhibiting the glycosphingolipid biosynthesis led to strong reduction of insulin secretion triggered by glucose or KCl, whereas inhibiting earlier steps of de novo ceramide synthesis resulted in milder inhibitory effect on insulin secretion by ND islets. Our data suggest that circadian clocks operative in human pancreatic islets are required for temporal orchestration of lipid homeostasis, and that perturbation of temporal regulation of the islet lipid metabolism upon T2D leads to altered insulin secretion and membrane fluidity. These phenotypes were recapitulated in ND islets bearing disrupted clocks.

Circulating 1,5-Anhydroglucitol as a Biomarker of ß-cell Mass Independent of a Diabetes Phenotype in Human Subjects.

CONTEXT: During an asymptomatic prediabetic state, the functional ß-cell mass decreases to a critical threshold, triggering diabetes and related symptoms. To date, there are no reliable readouts able to capture in vivo a potential drop of the ß-cell mass. OBJECTIVE: Beside its use as a short-term marker of glycemic control, the deoxyhexose 1,5-anhydroglucitol was identified in rodents as a circulating biomarker of the functional ß-cell mass already in the asymptomatic prediabetic stage. The present study investigated the putative corresponding relevance of circulating 1,5-anhydroglucitol in different human cohorts. METHODS: We analyzed clinical and blood parameters in patients with established type 2 diabetes and subjects considered at high risk of developing diabetes, as well as patients with no history of diabetes scheduled for pancreaticoduodenectomy. RESULTS: Circulating 1,5-anhydroglucitol was reduced in type 2 diabetic patients, negatively correlating with fasting plasma glucose (P < 0.0001) and hemoglobin A1c (P < 0.0001). In healthy subjects, 1,5-AG levels positively correlated with body mass index (P = 0.004) and Homeostatic Model Assessment of Insulin Resistance %S (P < 0.03) and was particularly high in nondiabetic obese individuals, potentially accounting for compensatory ß-cell expansion. Patients with no history of diabetes undergoing pancreaticoduodenectomy exhibited a 50% reduction of circulating 1,5-anhydroglucitol levels following surgery leading to an acute loss of their ß-cell mass (P = 0.002), regardless their glucose tolerance status. CONCLUSION: In summary, plasma concentration of 1,5-anhydroglucitol follows the ß-cell mass and its noninvasive monitoring may alert about the loss of ß cells in subjects at risk for diabetes, an event that cannot be captured by other clinical parameters of glycemic control.

Sex-specific modulation of circulating growth differentiation factor-15 in patients with type 2 diabetes and/or obesity.

Objective: Growth differentiation factor-15 (GDF15), a key metabolic regulator, is associated with obesity and diabetes in which sex-specific differences have been reported. Thus, we assessed whether GDF15 could be dependent on sex in diabetes and/or obesity groups. Methods: We measured serum GDF15 levels by ELISA in eight lean women and men (n = 16), eight women and eight men having obesity (n = 16), eight women and eight men with type 2 diabetes (T2D, n = 16), and seven women and nine men with both diabetes and obesity (n = 16). Estimation of the difference in the means of each group was performed by two-way ANOVA. The interdependence of the different variates was addressed by multivariate analysis. Correlations between GDF15 levels and HOMA-IR, HbA1c, triglycerides, HDL, and LDL were explored by linear regression. Results: Being a woman and having obesity alone or in combination with diabetes decreased GDF15 serum levels (ß = -0.47, CI = -0.95, 0.00, P = 0.052; ß = -0.45, CI = -0.94, 0.05, P= 0.075). Diabetes independently of metformin treatment and obesity were not predictive of low GDF15 levels (ß = 0.10, CI = -0.36, 0.57, P = 0.7). Correlation analysis showed that HOMA-IR (r = 0.45, P = 0.008) and triglycerides (r = 0.41, P = 0.017) were positively correlated and HDL (r = -0.48, P = 0.005) was negatively correlated with GDF15 levels in men. Conclusions/interpretation: GDF15 level was significantly different between men and women, as well as between the groups. Sex and group interaction revealed that being a woman and having obesity alone or in combination with diabetes decreased GDF15 levels.

Circadian rhythm of lipid metabolism.

Lipids comprise a diverse group of metabolites that are indispensable as energy storage molecules, cellular membrane components and mediators of inter- and intra-cellular signaling processes. Lipid homeostasis plays a crucial role in maintaining metabolic health in mammals including human beings. A growing body of evidence suggests that the circadian clock system ensures temporal orchestration of lipid homeostasis, and that perturbation of such diurnal regulation leads to the development of metabolic disorders comprising obesity and type 2 diabetes. In view of the emerging role of circadian regulation in maintaining lipid homeostasis, in this review, we summarize the current knowledge on lipid metabolic pathways controlled by the mammalian circadian system. Furthermore, we review the emerging connection between the development of human metabolic diseases and changes in lipid metabolites that belong to major classes of lipids. Finally, we highlight the mechanisms underlying circadian organization of lipid metabolic rhythms upon the physiological situation, and the consequences of circadian clock dysfunction for dysregulation of lipid metabolism.

The Effects of Shift Work on Cardio-Metabolic Diseases and Eating Patterns.

Energy metabolism is tightly linked with circadian rhythms, exposure to ambient light, sleep/wake, fasting/eating, and rest/activity cycles. External factors, such as shift work, lead to a disruption of these rhythms, often called circadian misalignment. Circadian misalignment has an impact on some physiological markers. However, these proxy measurements do not immediately translate into major clinical health outcomes, as shown by later detrimental health effects of shift work and cardio-metabolic disorders. This review focuses on the effects of shift work on circadian rhythms and its implications in cardio-metabolic disorders and eating patterns. Shift work appears to be a risk factor of overweight, obesity, type 2 diabetes, elevated blood pressure, and the metabolic syndrome. However, past studies showed discordant findings regarding the changes of lipid profile and eating patterns. Most studies were either small and short lab studies, or bigger and longer cohort studies, which could not measure health outcomes in a detailed manner. These two designs explain the heterogeneity of shift schedules, occupations, sample size, and methods across studies. Given the burden of non-communicable diseases and the growing concerns about shift workers' health, novel approaches to study shift work in real contexts are needed and would allow a better understanding of the interlocked risk factors and potential mechanisms involved in the onset of metabolic disorders.

Circadian clocks guide dendritic cells into skin lymphatics.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

Circadian hepatocyte clocks keep synchrony in the absence of a master pacemaker in the suprachiasmatic nucleus or other extrahepatic clocks.

It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.

The GLP-1R agonist liraglutide limits hepatic lipotoxicity and inflammatory response in mice fed a methionine-choline deficient diet.

Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder related to type 2 diabetes (T2D). The disease can evolve toward nonalcoholic steatohepatitis (NASH), a state of hepatic inflammation and fibrosis. There is presently no drug that effectively improves and/or prevents NAFLD/NASH/fibrosis. GLP-1 receptor agonists (GLP-1Ra) are effective in treating T2D. As with the endogenous gut incretins, GLP-1Ra potentiate glucose-induced insulin secretion. In addition, GLP-1Ra limit food intake and weight gain, additional beneficial properties in the context of obesity/insulin-resistance. Nevertheless, these pleiotropic effects of GLP-1Ra complicate the elucidation of their direct action on the liver. In the present study, we used the classical methionine-choline deficient (MCD) dietary model to investigate the potential direct hepatic actions of the GLP-1Ra liraglutide. A 4-week infusion of liraglutide (570 µg/kg/day) did not impact body weight, fat accretion or glycemic control in MCD-diet fed mice, confirming the suitability of this model for avoiding confounding factors. Liraglutide treatment did not prevent lipid deposition in the liver of MCD-fed mice but limited the accumulation of C16 and C24-ceramide/sphingomyelin species. In addition, liraglutide treatment alleviated hepatic inflammation (in particular accumulation of M1 pro-inflammatory macrophages) and initiation of fibrosis. Liraglutide also influenced the composition of gut microbiota induced by the MCD-diet. This included recovery of a normal Bacteroides proportion and, among the Erysipelotrichaceae family, a shift between Allobaculum and Turicibacter genera. In conclusion, liraglutide prevents accumulation of C16 and C24-ceramides/sphingomyelins species, inflammation and initiation of fibrosis in MCD-diet-fed mice liver, suggesting beneficial hepatic actions independent of weight loss and global hepatic steatosis.

Ether lipids, sphingolipids and toxic 1-deoxyceramides as hallmarks for lean and obese type 2 diabetic patients.

AIM: The worldwide increase in obesity and type 2 diabetes (T2D) represents a major health challenge. Chronically altered lipids induced by obesity further promote the development of T2D, and the accumulation of toxic lipid metabolites in serum and peripheral organs may contribute to the diabetic phenotype. METHODS: To better understand the complex metabolic pattern of lean and obese T2D and non-T2D individuals, we analysed the lipid profile of human serum, skeletal muscle and visceral adipose tissue of two cohorts by systematic mass spectrometry-based lipid analysis. RESULTS: Lipid homeostasis was strongly altered in a disease- and tissue-specific manner, allowing us to define T2D signatures associated with obesity from those that were obesity independent. Lipid changes encompassed lyso-, diacyl- and ether-phospholipids. Moreover, strong changes in sphingolipids included cytotoxic 1-deoxyceramide accumulation in a disease-specific manner in serum and visceral adipose tissue. The high amounts of non-canonical 1-deoxyceramide present in human adipose tissue most likely come from cell-autonomous synthesis because 1-deoxyceramide production increased upon differentiation to adipocytes in mouse cell culture experiments. CONCLUSION: Taken together, the observed lipidome changes in obesity and T2D will facilitate the identification of T2D patient subgroups and represent an important step towards personalized medicine in diabetes.

Circadian Lipidomics: Analysis of Lipid Metabolites Around the Clock.

Lipidomics has been defined as the large-scale analysis of lipids in organelles, cells, tissues, or whole organisms. Including the temporal aspects of lipid metabolic changes into this analysis allows to access yet another important aspect of lipid regulation. The resulting methodology, circadian lipidomics, has thus emerged as a novel tool to address the enormous complexity, which is present among cellular lipids. Here, we describe how mass spectrometry-based circadian lipidomics can be applied to study the impact of peripheral clocks on lipid metabolism in human primary cells and tissues, exemplified by studies in human pancreatic islets and skeletal myotubes.

Proinflammatory Cytokines Perturb Mouse and Human Pancreatic Islet Circadian Rhythmicity and Induce Uncoordinated ß-Cell Clock Gene Expression via Nitric Oxide, Lysine Deacetylases, and Immunoproteasomal Activity.

Pancreatic ß-cell-specific clock knockout mice develop ß-cell oxidative-stress and failure, as well as glucose-intolerance. How inflammatory stress affects the cellular clock is under-investigated. Real-time recording of Per2:luciferase reporter activity in murine and human pancreatic islets demonstrated that the proinflammatory cytokine interleukin-1ß (IL-1ß) lengthened the circadian period. qPCR-profiling of core clock gene expression in insulin-producing cells suggested that the combination of the proinflammatory cytokines IL-1ß and interferon-γ (IFN-γ) caused pronounced but uncoordinated increases in mRNA levels of multiple core clock genes, in particular of reverse-erythroblastosis virus α (Rev-erbα), in a dose- and time-dependent manner. The REV-ERBα/ß agonist SR9009, used to mimic cytokine-mediated Rev-erbα induction, reduced constitutive and cytokine-induced brain and muscle arnt-like 1 (Bmal1) mRNA levels in INS-1 cells as expected. SR9009 induced reactive oxygen species (ROS), reduced insulin-1/2 (Ins-1/2) mRNA and accumulated- and glucose-stimulated insulin secretion, reduced cell viability, and increased apoptosis levels, reminiscent of cytokine toxicity. In contrast, low (<5,0 µM) concentrations of SR9009 increased Ins-1 mRNA and accumulated insulin-secretion without affecting INS-1 cell viability, mirroring low-concentration IL-1ß mediated ß-cell stimulation. Inhibiting nitric oxide (NO) synthesis, the lysine deacetylase HDAC3 and the immunoproteasome reduced cytokine-mediated increases in clock gene expression. In conclusion, the cytokine-combination perturbed the intrinsic clocks operative in mouse and human pancreatic islets and induced uncoordinated clock gene expression in INS-1 cells, the latter effect associated with NO, HDAC3, and immunoproteasome activity.

The core clock transcription factor BMAL1 drives circadian ß-cell proliferation during compensatory regeneration of the endocrine pancreas.

Circadian clocks in pancreatic islets participate in the regulation of glucose homeostasis. Here we examined the role of these timekeepers in ß-cell regeneration after the massive ablation of ß cells by doxycycline-induced expression of diphtheria toxin A (DTA) in Insulin-rtTA/TET-DTA mice. Since we crossed reporter genes expressing α- and ß-cell-specific fluorescent proteins into these mice, we could follow the fate of α- and ß cells separately. As expected, DTA induction resulted in an acute hyperglycemia, which was accompanied by dramatic changes in gene expression in residual ß cells. In contrast, only temporal alterations of gene expression were observed in α cells. Interestingly, ß cells entered S phase preferentially during the nocturnal activity phase, indicating that the diurnal rhythm also plays a role in the orchestration of ß-cell regeneration. Indeed, in arrhythmic Bmal1-deficient mice, which lack circadian clocks, no compensatory ß-cell proliferation was observed, and the ß-cell ablation led to aggravated hyperglycemia, hyperglucagonemia, and fatal diabetes.

Coupled network of the circadian clocks: a driving force of rhythmic physiology.

The circadian system is composed of coupled endogenous oscillators that allow living beings, including humans, to anticipate and adapt to daily changes in their environment. In mammals, circadian clocks form a hierarchically organized network with a 'master clock' located in the suprachiasmatic nucleus of the hypothalamus, which ensures entrainment of subsidiary oscillators to environmental cycles. Robust rhythmicity of body clocks is indispensable for temporally coordinating organ functions, and the disruption or misalignment of circadian rhythms caused for instance by modern lifestyle is strongly associated with various widespread diseases. This review aims to provide a comprehensive overview of our current knowledge about the molecular architecture and system-level organization of mammalian circadian oscillators. Furthermore, we discuss the regulatory roles of peripheral clocks for cell and organ physiology and their implication in the temporal coordination of metabolism in human health and disease. Finally, we summarize methods for assessing circadian rhythmicity in humans.