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Type 2 diabetes mellitus (T2DM) is characterized by β-cell dysfunction as a result of impaired glucose-stimulated insulin secretion (GSIS). Studies show that β-cell circadian clocks are important regulators of GSIS and glucose homeostasis. These observations raise the question about whether enhancement of the circadian clock in β-cells will confer protection against β-cell dysfunction under diabetogenic conditions. To test this, we used an approach by first generating mice with β-cell–specific inducible overexpression of Bmal1 (core circadian transcription factor; β-Bmal1OV). We subsequently examined the effects of β-Bmal1OV on the circadian clock, GSIS, islet transcriptome, and glucose metabolism in the context of diet-induced obesity. We also tested the effects of circadian clock–enhancing small-molecule nobiletin on GSIS in mouse and human control and T2DM islets. We report that β-Bmal1OV mice display enhanced islet circadian clock amplitude and augmented in vivo and in vitro GSIS and are protected against obesity-induced glucose intolerance. These effects were associated with increased expression of purported BMAL1-target genes mediating insulin secretion, processing, and lipid metabolism. Furthermore, exposure of isolated islets to nobiletin enhanced β-cell secretory function in a Bmal1-dependent manner. This work suggests therapeutic targeting of the circadian system as a potential strategy to counteract β-cell failure under diabetogenic conditions.

BACKGROUND The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), has emerged as a major pest of cotton, Gossypium hirsutum L, in the mid-southern USA. In the early 1990s L. lineolaris populations developed resistance to several classes of conventional insecticides, increasing the need for insecticides with alternative modes of action such as insect growth regulators (IGRs) for integrated pest management (IPM). The benzoylphenyl urea (BPU) class of IGRs acts by disrupting the growth and development of immature stages of insects, but little is known about its impact on adult stages. RESULTS The effect of novaluron (Diamond™ 0.83EC), a BPU with known chitin synthesis inhibitor activity, was investigated on adult females of L. lineolaris. Treatment of 1-day-old adults with 600 ppm of novaluron in the diet prevented oviposition, while treatment of older females had no impact on oviposition. Oral novaluron exposure of adults of all ages reduced the viability of eggs laid. Novaluron treatment caused ultrastructural changes in the ovaries of 1-day-old adults (48 h post exposure), distorting the follicular epithelial cell architecture of developing oocytes. Additionally, novaluron treatment decreased the chitin content in ovarian tissue. CONCLUSION Our results suggest that chitin or chitin-like components in the developing ovaries of adult L. lineolaris are a target of IGRs such as novaluron, but its activity is specific to a critical time during development. This enhances our understanding of the effects of BPUs on adult insects and could lead to incorporation of IGRs in IPM for controlling adult insect pest populations in the field. © 2020 Society of Chemical Industry.

Pancreatic β cell failure in type 2 diabetes mellitus (T2DM) is attributed to perturbations of the β cell’s transcriptional landscape resulting in impaired glucose-stimulated insulin secretion. Recent studies identified SLC4A4 (a gene encoding an electrogenic Na(+)-coupled HCO(3)(–) cotransporter and intracellular pH regulator, NBCe1) as one of the misexpressed genes in β cells of patients with T2DM. Thus, in the current study, we set out to test the hypothesis that misexpression of SLC4A4/NBCe1 in T2DM β cells contributes to β cell dysfunction and impaired glucose homeostasis. To address this hypothesis, we first confirmed induction of SLC4A4/NBCe1 expression in β cells of patients with T2DM and demonstrated that its expression was associated with loss of β cell transcriptional identity, intracellular alkalinization, and β cell dysfunction. In addition, we generated a β cell–selective Slc4a4/NBCe1-KO mouse model and found that these mice were protected from diet-induced metabolic stress and β cell dysfunction. Importantly, improved glucose tolerance and enhanced β cell function in Slc4a4/NBCe1-deficient mice were due to augmented mitochondrial function and increased expression of genes regulating β cell identity and function. These results suggest that increased β cell expression of SLC4A4/NBCe1 in T2DM plays a contributory role in promotion of β cell failure and should be considered as a potential therapeutic target.

SUMMARY 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 β cell communication and function. Compelling evidence has implicated extracellular vesicles (EVs) in modulating physiological and pathological responses to β cell stress. We report that pro-inflammatory β cell small EVs (cytokine-exposed EVs [cytoEVs]) induce β 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 β cells. CXCR3 receptor inhibition reduced CXCL10-cytoEV binding and attenuated β cell dysfunction, inflammatory gene expression, and leukocyte recruitment to islets. This work implies a significant role of pro-inflammatory β cell-derived small EVs in modulating β cell function, global gene expression, and antigen presentation through activation of the CXCL10/CXCR3 axis., Graphical Abstract, In brief Javeed et al. demonstrate the importance of pro-inflammatory-exposed β cell-derived extracellular vesicles (cytoEVs) as facilitators of β cell dysfunction and a pro-inflammatory islet microenvironment. Physiological and mechanistic evidence implicates CXCL10/CXCR3 axis activation mediated by cytoEVs and improvements in β cell function upon CXCR3 receptor blockade.

Obesity is associated with several chronic comorbidities, one of which is type 2 diabetes mellitus (T2DM). The pathogenesis of obesity and T2DM is influenced by alterations in diet macronutrient composition, which regulate energy expenditure, metabolic function, glucose homeostasis, and pancreatic islet cell biology. Recent studies suggest that increased intake of dietary carbohydrates plays a previously underappreciated role in the promotion of obesity and consequent metabolic dysfunction. Thus, in this study, we utilized mouse models to test the hypothesis that dietary carbohydrates modulate energetic, metabolic, and islet adaptions to high-fat diets. To address this, we exposed C57BL/6J mice to 12 wk of 3 eucaloric high-fat diets (>60% calories from fat) with varying total carbohydrate (1–20%) and sucrose (0–20%) content. Our results show that severe restriction of dietary carbohydrates characteristic of ketogenic diets reduces body fat accumulation, enhances energy expenditure, and reduces prevailing glycemia and insulin resistance compared with carbohydrate-rich, high-fat diets. Moreover, severe restriction of dietary carbohydrates also results in functional, morphological, and molecular changes in pancreatic islets highlighted by restricted capacity for β-cell mass expansion and alterations in insulin secretory response. These studies support the hypothesis that low-carbohydrate/high-fat diets provide antiobesogenic benefits and suggest further evaluation of the effects of these diets on β-cell biology in humans.

The majority of patients with pancreatic ductal adenocarcinoma (PC) display either impaired fasting glucose/glucose intolerance or overt diabetes. However, the pathophysiologic basis of this association remains largely unexplained.In this case-control study we aimed to study the morphological changes in the islets of patients with PC, compared to control patients with and without type 2 diabetes mellitus (T2DM). T2DM controls and PC cases had a lower β-cell area and average islet size and density compared to non-T2DM controls (p 0.05).Compared to both T2DM and non-T2DM controls, mean α-cell area was significantly lower and β/α-ratio was higher in PC cases (p 0.05). Furthermore, whereas islets in T2DM controls were characterized by disrupted islet architecture and presence of islet amyloid aggregates, islet composition in PC islets was not significantly different compared to non-T2DM controls (p 0.05 vs. Control).Our data shows that PC is associated with a unique pattern of islet pathology characterized by preserved architecture, absence of amyloid aggregates, and relative α-cell loss indicating that distinct mechanisms are likely involved in the pathophysiology of islet failure in PC-induced DM. Insights into the mechanisms mediating β-cell failure in PC can be important for our understanding of pathophysiology of PC.

The worldwide prevalence of type 2 diabetes (T2D) is increasing. Despite normal to higher bone density, patients with T2D paradoxically have elevated fracture risk resulting, in part, from poor bone quality. Advanced glycation endproducts (AGEs) and inflammation as a consequence of enhanced receptor for AGE (RAGE) signaling are hypothesized culprits, although the exact mechanisms underlying skeletal dysfunction in T2D are unclear. Lack of inducible models that permit environmental (in obesity) and temporal (after skeletal maturity) control of T2D onset has hampered progress. Here, we show in C57BL/6 mice that a onetime pharmacological intervention (streptozotocin, STZ) initiated in adulthood combined with high-fat diet-induced (HFD-induced) obesity caused hallmark features of human adult-onset T2D, including prolonged hyperglycemia, insulin resistance, and pancreatic β cell dysfunction, but not complete destruction. In addition, HFD/STZ (i.e., T2D) resulted in several changes in bone quality that closely mirror those observed in humans, including compromised bone microarchitecture, reduced biomechanical strength, impaired bone material properties, altered bone turnover, and elevated levels of the AGE CML in bone and blood. Furthermore, T2D led to the premature accumulation of senescent osteocytes with a unique proinflammatory signature. These findings highlight the RAGE pathway and senescent cells as potential targets to treat diabetic skeletal fragility.

Development of cell replacement therapies in diabetes requires understanding of the molecular underpinnings of β-cell maturation. The circadian clock regulates diverse cellular functions important for regulation of β-cell function and turnover. However, postnatal ontogenesis of the islet circadian clock and its potential role in β-cell maturation remain unknown. To address this, we studied wild-type Sprague-Dawley as well as Period1 luciferase transgenic (Per1:LUC) rats to determine circadian clock function, clock protein expression, and diurnal insulin secretion during islet development and maturation process. We additionally studied β-cell–specific Bmal1-deficient mice to elucidate a potential role of this key circadian transcription factor in β-cell functional and transcriptional maturation. We report that emergence of the islet circadian clock 1) occurs during the early postnatal period, 2) depends on the establishment of global behavioral circadian rhythms, and 3) leads to the induction of diurnal insulin secretion and gene expression. Islet cell maturation was also characterized by induction in the expression of circadian transcription factor BMAL1, deletion of which altered postnatal development of glucose-stimulated insulin secretion and the associated transcriptional network. Postnatal development of the islet circadian clock contributes to early-life β-cell maturation and should be considered for optimal design of future β-cell replacement strategies in diabetes.

Intrinsic β-cell circadian clocks are a prerequisite for the control of glucose homeostasis through regulation of β-cell function and turnover. However, little is known about the contributions of circadian clock disruption to the natural progression of β-cell failure in diabetes. To address this, we examined the effects of cytokine-mediated inflammation, common to the pathophysiology of Type 1 and Type 2 diabetes, on the physiological, molecular, and epigenetic regulation of circadian clocks in β-cells. Specifically, we provide evidence that the key diabetogenic cytokine IL-1β disrupts functionality of the β-cell circadian clock and circadian regulation of insulin secretion through impaired expression of the key transcription factor Bmal1, evident at the level of promoter activation, mRNA, and protein expression. Additionally, IL-1β-mediated inflammation was shown to augment genome-wide DNA-binding patterns ofBmal1(and its heterodimer,Clock) in β-cells towards binding sites in the proximity of genes annotated to pathways regulating β-cell apoptosis, inflammation, and dedifferentiation. Finally, we identified that the development of hyperglycemia in humans is associated with compromised β-cell BMAL1 expression suggestive of a causative link between circadian clock disruption and β-cell failure in diabetes.

Pancreatic islet failure is a characteristic feature of impaired glucose control in diabetes mellitus. Circadian control of islet function is essential for maintaining proper glucose homeostasis. Circadian variations in transcriptional pathways have been described in diverse cell types and shown to be critical for optimization of cellular function in vivo. In the current study, we utilized Short Time Series Expression Miner (STEM) analysis to identify diurnally expressed transcripts and biological pathways from mouse islets isolated at 4 h intervals throughout the 24 h light-dark cycle. STEM analysis identified 19 distinct chronological model profiles, and genes belonging to each profile were subsequently annotated to significantly enriched Kyoto Encyclopedia of Genes and Genomes biological pathways. Several transcriptional pathways essential for proper islet function (e.g., insulin secretion, oxidative phosphorylation), cell survival (e.g., insulin signaling, apoptosis) and cell proliferation (DNA replication, homologous recombination) demonstrated significant time-dependent variations. Notably, KEGG pathway analysis revealed “protein processing in endoplasmic reticulum - mmu04141” as one of the most enriched time-dependent pathways in islets. This study provides unique data set on time-dependent diurnal profiles of islet gene expression and biological pathways, and suggests that diurnal variation of the islet transcriptome is an important feature of islet homeostasis, which should be taken into consideration for optimal experimental design and interpretation of future islet studies.

Endogenous circadian clocks orchestrate several metabolic and signaling pathways that are known to modulate lifespan, suggesting clocks as potential targets for manipulation of metabolism and lifespan. We report here that the core circadian clock genes, timeless (tim) and period (per), are required for the metabolic and lifespan responses to DR in Drosophila. Consistent with the involvement of a circadian mechanism, DR enhances the amplitude of cycling of most circadian clock genes, including tim, in peripheral tissues. Mass-spectrometry-based lipidomic analysis suggests a role of tim in cycling of specific medium chain triglycerides under DR. Furthermore, overexpression of tim in peripheral tissues improves its oscillatory amplitude and extends lifespan under ad libitum conditions. Importantly, effects of tim on lifespan appear to be mediated through enhanced fat turnover. These findings identify a critical role for specific clock genes in modulating the effects of nutrient manipulation on fat metabolism and aging.

This study was focused on gaining insights into the mechanism by which the herbicide- Spectracide®, induces oxidative stress and alters behavior in Drosophila melanogaster. Exposure to Spectracide® (50%) significantly (p 0.05) reduced the negative geotaxis response, jumping behavior and dampened locomotor activity rhythm in adult flies compared to non-exposed flies. Protein carbonyl levels indicative of oxidative damage increased significantly coupled with down-regulation of Sniffer gene expression encoding carbonyl reductase (CR) and its activity in Spectracide®-exposed flies. In silico modeling analysis revealed that the active ingredients of Spectracide® (atrazine, diquat dibromide, fluazifop-p-butyl, and dicamba) have significant binding affinity to the active site of CR enzyme, with atrazine having comparatively greater affinity. Our results suggest a mechanism by which ingredients in Spectracide® induce oxidative damage by competitive binding to the active site of a protective enzyme and impair its ability to prevent damage to proteins thereby leading to deficits in locomotor behavior in Drosophila.

Both type 1 and type 2 diabetes are characterized by a decline in β-cell function and mass partly attributed to exposure of β-cells to proinflammatory cytokines. The molecular mechanisms underlying this process are not well understood, however several lines of evidence have linked the circadian clock with regulation of the deleterious proinflammatory effects in diabetes. Therefore, in this study, we assessed the effect of known proinflammatory mediators of β-cell failure in diabetes (IL-1β, TNFα, IL-6, and IFNγ) on the β-cell circadian clock. Through real-time bioluminescence tracking of islets from Per2:luciferase (a key circadian clock gene reporter) knock-in mice, our data revealed that IL-1β (2 ng/ml) diminished β-cell circadian rhythmicity via impairment of the amplitude and the phase of oscillations (p Disclosure N. Javeed: None. K. Rakshit: None. A. Matveyenko: None.

Summary Generation of functional β cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However, it has been challenging to generate competent β cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors, critical for β-cell development and function, in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived β-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1, NEUROG3, and MAFA triad (PNM) enabled in vitro generation of psBCs with glucose and GLP-1 responsiveness within 3 weeks. PNM transduction upregulated genes associated with glucose sensing, insulin secretion, and β-cell maturation. In recipient diabetic mice, PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1 week post transplantation. Thus, enhanced pre-emptive β-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in vitro, offering a competent tissue-primed biotherapy., Highlights • PNM transduction facilitates generation of glucose- and GLP1-responsive β cells • Stepwise PNM improves glucose sensing, insulin secretion, and β-cell maturation • PNM-transduced psBCs showed glucose-responsive insulin secretion in diabetic mice, In this article, Ikeda Yasuhiro and colleagues show that stepwise transduction of the triad of transcription factors PDX1, NEUROG3, and MAFA (PNM) enabled in vitro generation of glucose- and GLP-1-responsive β cells from iPSCs within 3 weeks. PNM transduction improves glucose sensing, insulin secretion, and β-cell maturation of generated cells. PNM-transduced β cells showed glucose-responsive insulin secretion as early as 1 week post transplantation in diabetic mice.

High-fat diet (HFD)-fed mouse models have been widely used to study early type 2 diabetes. Decreased β-cell glucokinase (GCK) expression has been observed in HFD-induced diabetes. However, owing to its crucial roles in glucose metabolism in the liver and in islet β-cells, the contribution of decreased GCK expression to the development of HFD-induced diabetes is unclear. Here, we employed a β-cell-targeted gene transfer vector and determined the impact of β-cell-specific increase in GCK expression on β-cell function and glucose handling in vitro and in vivo. Overexpression of GCK enhanced glycolytic flux, ATP-sensitive potassium channel activation and membrane depolarization, and increased proliferation in Min6 cells. β-cell-targeted GCK transduction did not change glucose handling in chow-fed C57BL/6 mice. Although adult mice fed a HFD showed reduced islet GCK expression, impaired glucose tolerance and decreased glucose-stimulated insulin secretion (GSIS), β-cell-targeted GCK transduction improved glucose tolerance and restored GSIS. Islet perifusion experiments verified restored GSIS in isolated HFD islets by GCK transduction. Thus, our data identify impaired β-cell GCK expression as an underlying mechanism for dysregulated β-cell function and glycemic control in HFD-induced diabetes. Our data also imply an etiological role of GCK in diet-induced diabetes. This article has an associated First Person interview with the first author of the paper., Summary: β-cell glucokinase expression is decreased in diet-induced diabetes. β-cell-targeted overexpression of glucokinase improved the diabetic phenotype, suggesting an etiological role of glucokinase downregulation in diet-induced diabetes.

There are clear epidemiological associations between circadian disruption, obesity, and pathogenesis of type 2 diabetes. The mechanisms driving these associations are unclear. In the current study, we hypothesized that continuous exposure to constant light (LL) compromises pancreatic β-cell functional and morphological adaption to diet-induced obesity leading to development of type 2 diabetes. To address this hypothesis, we studied wild type Sprague Dawley as well as Period-1 luciferase reporter transgenic rats (Per1-Luc) for 10 weeks under standard light-dark cycle (LD) or LL with concomitant ad libitum access to either standard chow or 60% high-fat diet (HFD). Exposure to HFD led to a comparable increase in food intake, body weight, and adiposity in both LD- and LL-treated rats. However, LL rats displayed profound loss of behavioral circadian rhythms as well as disrupted pancreatic islet clock function characterized by the impairment in the amplitude and the phase islet clock oscillations. Under LD cycle, HFD did not adversely alter diurnal glycemia, diurnal insulinemia, β-cell secretory function as well as β-cell survival, indicating successful adaptation to increased metabolic demand. In contrast, concomitant exposure to LL and HFD resulted in development of hyperglycemia characterized by loss of diurnal changes in insulin secretion, compromised β-cell function, and induction of β-cell apoptosis. This study suggests that circadian disruption and diet-induced obesity synergize to promote development of β-cell failure, likely mediated as a consequence of impaired islet clock function.

The impact of mutations in four essential genes involved in dopamine (DA) synthesis and transport on longevity, motor behavior, and resistance to oxidative stress was monitored in Drosophila melanogaster. The fly lines used for this study were: (i) a loss of function mutation in Catecholamines up (Catsup(26)), which is a negative regulator of the rate limiting enzyme for DA synthesis, (ii) a mutant for the gene pale (ple(2)) that encodes for the rate limiting enzyme tyrosine hydroxylase (TH), (iii) a mutant for the gene Punch (Pu(Z22)) that encodes guanosine triphosphate cyclohydrolase, required for TH activity, and (iv) a mutant in the vesicular monoamine transporter (VMAT(Δ14)), which is required for packaging of DA as vesicles inside DA neurons. Median lifespans of ple(2), Pu(Z22) and VMAT(Δ14) mutants were significantly decreased compared to Catsup(26) and wild type controls that did not significantly differ between each other. Catsup(26) flies survived longer when exposed to hydrogen peroxide (80 μM) or paraquat (10mM) compared to ple(2), Pu(Z22) or VMAT(Δ14) and controls. These flies also exhibited significantly higher negative geotaxis activity compared to ple(2), Pu(Z22), VMAT(Δ14) and controls. All mutant flies demonstrated rhythmic circadian locomotor activity in general, albeit Catsup(26) and VMAT(Δ14) flies had slightly weaker rhythms. Expression analysis of some key antioxidant genes revealed that glutathione S-transferase Omega-1 (GSTO1) expression was significantly up-regulated in all DA synthesis pathway mutants and especially in Catsup(26) and VMAT(Δ14) flies at both mRNA and protein levels. Taken together, we hypothesize that DA could directly influence GSTO1 transcription and thus play a significant role in the regulation of response to oxidative stress. Additionally, perturbations in DA synthesis do not appear to have a significant impact on circadian locomotor activity rhythms per se, but do have an influence on general locomotor activity levels.

Daily life functions such as sleep and feeding oscillate with circa 24 h period due to endogenous circadian rhythms generated by circadian clocks. Genetic or environmental disruption of circadian rhythms is associated with various aging-related phenotypes. Circadian rhythms decay during normal aging, and there is a need to explore strategies that could avert age-related changes in the circadian system. Exercise was reported to delay aging in mammals. Here, we investigated whether daily exercise via stimulation of upward climbing movement could improve circadian rest/activity rhythms in aging Drosophila melanogaster. We found that repeated exercise regimen did not strengthen circadian locomotor activity rhythms in aging flies and had no effect on their lifespan. We also tested the effects of exercise on mobility and determined that regular exercise lowered age-specific climbing ability in both wild type and clock mutant flies. Interestingly, the climbing ability was most significantly reduced in flies carrying a null mutation in the core clock gene period, while rescue of this gene significantly improved climbing to wild type levels. Our work highlights the importance of period in sustaining endurance in aging flies exposed to physical challenge.

Circadian clocks generate daily rhythms in molecular, cellular, and physiological functions providing temporal dimension to organismal homeostasis. Recent evidence suggests two-way relationship between circadian clocks and aging. While disruption of the circadian clock leads to premature aging in animals, there is also age-related dampening of output rhythms such as sleep/wake cycles and hormonal fluctuations. Decay in the oscillations of several clock genes was recently reported in aged fruit flies, but mechanisms underlying these age-related changes are not understood. We report that the circadian light-sensitive protein CRYPTOCHROME (CRY), is significantly reduced at both mRNA and protein levels in heads of old Drosophila melanogaster. Restoration of CRY using the binary GAL4/UAS system in old flies significantly enhanced the mRNA oscillatory amplitude of several genes involved in the clock mechanism. Flies with CRY overexpressed in all clock cells maintained strong rest/activity rhythms in constant darkness late in life when rhythms were disrupted in most control flies. WE also observed a remarkable extension of healthspan in flies with elevated CRY. Conversely, CRY deficient mutants showed accelerated functional decline and accumulated greater oxidative damage. Interestingly, overexpression of CRY in central clock neurons alone was not sufficient to restore rest/activity rhythms or extend healthspan. Together, these data suggest novel anti-aging functions of CRY and indicate that peripheral clocks play an active role in delaying behavioral and physiological aging.

Circadian clocks maintain temporal homeostasis by generating daily output rhythms in molecular, cellular, and physiological functions. Output rhythms, such as sleep/wake cycles and hormonal fluctuations, tend to deteriorate during aging in humans, rodents, and fruit flies. However, it is not clear whether this decay is caused by defects in the core transcriptional clock, or weakening of the clock-output pathways, or both. The authors monitored age-related changes in behavioral and molecular rhythms in Drosophila melanogaster. Aging was associated with disrupted rest/activity patterns and lengthening of the free-running period of the circadian locomotor activity rhythm. The expression of core clock genes was measured in heads and bodies of young, middle-aged, and old flies. Transcriptional oscillations of four clock genes, period, timeless, Par domain protein 1ϵ, and vrille, were significantly reduced in heads, but not in bodies, of aging flies. It was determined that reduced transcription of these genes was not caused by the deficient expression of their activators, encoded by Clock and cycle genes. Interestingly, transcriptional activation by CLOCK-CYCLE complexes was impaired despite reduced levels of the PERIOD repressor protein in old flies. These data suggest that aging alters the properties of the core transcriptional clock in flies such that both the positive and the negative limbs of the clock are attenuated.

Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but whether the circadian clock regulates the adaptive immune response to cancer is mostly unclear. In mouse models of polyposis, and human colorectal cancer patients, we showed that tumor infiltrating Tregs overexpressed b-catenin and RORgt, and secreted IL-17. These Tregs suppressed tumor reactive cytotoxic T cells, and promoted tumor growth. Therefore, we examined how the expression of a dominant negative ClockΔ19 gene affects inflammation and susceptibility to cancer in the APCΔ468mouse model of hereditary polyposis. ClockΔ19 mutant mice had high expression of RORgt+ and b-catenin+ Tregs. These mice showed a notable expansion of Th17 inflammation in vivo upon injection of aCD3. Functionally these Tregs showed pro-inflammatory properties. In addition to these effects, APCΔ468ClockΔ19 mice had increased numbers of CD11b+ cell, Ly6C+CXCR1− monocytes, and Ly6C+Ly6G+ myeloid derived suppressor cells (MDSC) as compared with APCΔ468 mice. APCΔ468ClockΔ19 mice had higher tumor burden in the small intestine and colon than the APCΔ468 mice. Both T-cells and tumors had intense stabilization of nuclear b-catenin and were more aggressive as indicated by abundance of stromal mMCP6+ mast cells (MC), which typically appear at the invasive borders of tumors. Our finding indicating increase in tumor load and worsening inflammation caused by direct ClockΔ19 effects in the epithelium and enhanced TH17 bias in the immune system.

There is increasing evidence that aging is affected by biological (circadian) clocks - the internal mechanisms that coordinate daily changes in gene expression, physiological functions and behavior with external day/night cycles. Recent data suggest that disruption of the mammalian circadian clock results in accelerated aging and increased age-related pathologies such as cancer; however, the links between loss of daily rhythms and aging are not understood. We sought to determine whether disruption of the circadian clock affects lifespan and healthspan in the model organism Drosophila melanogaster. We examined effects of a null mutation in the circadian clock gene period (per(01)) on the fly healthspan by challenging aging flies with short-term oxidative stress (24h hyperoxia) and investigating their response in terms of mortality hazard, levels of oxidative damage, and functional senescence. Exposure to 24h hyperoxia during middle age significantly shortened the life expectancy in per(01) but not in control flies. This homeostatic challenge also led to significantly higher accumulation of oxidative damage in per(01) flies compared to controls. In addition, aging per(01) flies showed accelerated functional decline, such as lower climbing ability and increased neuronal degeneration compared to age-matched controls. Together, these data suggest that impaired stress defense pathways may contribute to accelerated aging in the per mutant. In addition, we show that the expression of per gene declines in old wild type flies, suggesting that the circadian regulatory network becomes impaired with age.

Circadian regulation of glucose homeostasis and insulin secretion has long been appreciated as an important feature of metabolic control in humans. Circadian disruption is becoming increasingly prevalent in today’s society and is likely responsible in part for the considerable rise in Type 2 diabetes (T2DM) and metabolic syndrome worldwide. Thus, understanding molecular mechanisms driving the inter-relationship between circadian disruption and T2DM is important in context of disease prevention and therapeutics. In this regard, the goal of this manuscript is to highlight the role of the circadian system, and islet circadian clocks in particular, as potential regulators of β-cell function and survival. To date, studies have shown that islet clocks respond to changes in feeding patterns, and regulate a multitude of critical cellular processes in insulin secreting β-cells (e.g. insulin exocytosis, mitochondrial function and response to oxidative stress). Subsequently, either genetic or environmental disruption of normal islet clock performance compromises β-cell function and leads to loss of glycemic control. Future work is warranted to further unravel the role of circadian clocks in human islet function in health and contributions to pathogenesis of T2DM.

Obesity and consequent insulin resistance are known risk factors for type 2 diabetes. A compensatory increase in beta cell function and mass in response to insulin resistance permits maintenance of normal glucose homeostasis, whereas failure to do so results in beta cell failure and type 2 diabetes. Recent evidence suggests that the circadian system is essential for proper metabolic control and regulation of beta cell function. We set out to address the hypothesis that the beta cell circadian clock is essential for the appropriate functional and morphological beta cell response to insulin resistance. We employed conditional deletion of the Bmal1 (also known as Arntl) gene (encoding a key circadian clock transcription factor) in beta cells using the tamoxifen-inducible CreERT recombination system. Upon adulthood, Bmal1 deletion in beta cells was achieved and mice were exposed to either chow or high fat diet (HFD). Changes in diurnal glycaemia, glucose tolerance and insulin secretion were longitudinally monitored in vivo and islet morphology and turnover assessed by immunofluorescence. Isolated islet experiments in vitro were performed to delineate changes in beta cell function and transcriptional regulation of cell proliferation. Adult Bmal1 deletion in beta cells resulted in failed metabolic adaptation to HFD characterised by fasting and diurnal hyperglycaemia, glucose intolerance and loss of glucose-stimulated insulin secretion. Importantly, HFD-induced beta cell expansion was absent following beta cell Bmal1 deletion indicating impaired beta cell proliferative and regenerative potential, which was confirmed by assessment of transcriptional profiles in isolated islets. Results of the study suggest that the beta cell circadian clock is a novel regulator of compensatory beta cell expansion and function in response to increased insulin demand associated with diet-induced obesity.

Circadian clocks are endogenous molecular regulators that coordinate daily changes in gene expression and cellular and physiological functions with external day/ night cycles. The core circadian clock in the fruit fly Drosophila melanogaster, consists of interacting molecular feedback loops. In the main loop, transcriptional activators (called positive clock elements), encoded by genes Clock (Clk) and cycle (cyc), stimulate the expression of two other clock genes, period (per) and timeless (tim), in the early night [1]. This leads to periodic increases in the levels of per/tim mRNA and PER/TIM proteins. These proteins (called negative elements of the clock) accumulate in cell nuclei late at night and act as inhibitors of the CLK-CYC complexes, resulting in the suppression of per and tim transcription (Fig. 23.1) [2]. In the second feedback loop, daily oscillations of Clk mRNA are achieved via a rhythmically active transcriptional repressor, encoded by vrille (vri), and transcriptional activator, encoded by Pdp1e [3–6]. Rhythmic expression of vri and Pdp1e is also activated by CLK-CYC complexes, via a similar mechanism as per and tim (Fig. 23.1). CLK is a basic helix-loop-helix (bHLH) transcription factor that has a PER-ARNT-SIM (PAS) protein interaction domain. An approximately 69-base (b) enhancer sequence is situated B500 b upstream of the per transcription start site (TSS). Within this enhancer, a consensus “E-box” bHLH transcription factor binding site having the sequence CACGTG is required for transcriptional activation [7]. Likewise, a consensus CACGTG E-box, approximately 2.5 kilobases (kb) upstream of tim, has been known to be essential for its transcriptional activation [8, 9]. Recent studies have shown strong rhythmic binding of CLK to the E-box upstream of per [10, 11]. Also. there was weak rhythmic binding of CLK to the intronic E-box of tim [11]. The circadian rhythms generated by these clocks are responsible for 24-h oscillations in diverse biological processes. While the central genes governing circadian pacemaker rhythmicity havemostly been identified, clockcontrolled output molecules responsible for regulating rhythmic behaviors remain largely unknown. Hence, identification of genes controlled by the central clock would be important in providing an avenue for understanding circadian oscillations in diverse biological processes including locomotor activity, feeding behavior, hormone secretion, digestion, and the onset of sleep. Genomewide studies of circadian gene expression revealed rhythms in the expression of multiple genes involved in various metabolic pathways and stress response in D. melanogaster [12–14]. A number of genes involved

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila.