Abstract Introduction Sleep deprivation, aseptic injury, and infection activate the innate immune response and promote recovery sleep, or “sickness” sleep. In Drosophila, two NFκB genes, Dif and Relish, are required for promoting sleep following sleep deprivation and infection, respectively. nemuri, a sleep promoting antimicrobial peptide, is also induced by sleep deprivation and infection, and dependent on Dif and Relish, respectively, for its induction. NFκBs contribute to synthesis of membrane phospholipids through an IRE1/xbp1-dependent mechanism, possibly due to lipid bilayer stress caused by massive induction of antimicrobial peptides. We have observed that upregulation of nemuri in the brain severely reduces lipid droplets throughout the fly. Using lipidomics we tested whether upregulation of nemuri disrupts lipid homeostasis and thereby promotes sickness sleep. Methods Female flies over expressing nemuri (elavC155-Gal4>UAS-nemuri) and parental controls (elavC155-Gal4/+ and +/UAS-nemuri) were collected at ZT 2 and 14. Flies from different time points were pooled to eliminate circadian effects. Lipids isolated from heads were subjected to lipid profiling (n=4 samples per group) using Desi-Mass spectrometry. Positive and negative mode lipidomics data were subjected to multivariate orthogonal partial least squares-discriminant analysis (OPLSDA). Multivariate analysis was used to guide the classification of samples. Individual lipids were compared using non-parametric tests. Results Untargeted high resolution mass spectrometric lipid profiling of these flies indicated significant increases in lipids detected in both positive and negative modes with glycerophospholipids and sphingolipids comprising over 60% of those affected. Other lipid types that were increased included acylcarnitines, fatty acids, and glycerolipids. Cardiolipins in particular were uniquely increased by upregulation of nemuri, suggesting a disruption of mitochondrial function. Reducing lipid bilayer stress reduced excessive sleep in nemuri over-expressing flies. Conclusion These findings indicate an effect of nemuri on lipid remodeling in plasma membranes. Addressing disruption of lipid homeostatic factors could resolve excess sleepiness or fatigue that occurs with chronic illness. Support (if any) NIH R01NS124698 "Interactions between the immune response and lipid homeostasis in regulating sleep during sickness"

Biological clocks shape metabolism, but how circadian programs govern nutrient processing is unclear. Here, using human metabolomics and 13C6-glucose tracing in Drosophila, we delineate previously under characterized daily oscillations in glucose-derived metabolic networks, providing a mechanistic framework for a purpose-built isotope-tracing approach. In flies, we reveal a pronounced "rush hour" of glucose utilization early in the light phase, with carbons directed to biosynthetic and energetic pathways. By contrast, a dopamine reuptake-deficient hyperactive mutant (fumin) with elevated metabolic rate shows phase-shifted and amplified metabolic peaks, indicating that altered neural signaling reshapes temporal glucose flux. Neither altered feeding schedules nor short-term fasting disrupt these intrinsic metabolic rhythms, strongly suggesting that circadian timing, rather than nutrient availability, orchestrates temporal homeostasis. By integrating human metabolite profiling with isotope-tracing in flies, we define a conserved temporal architecture of glucose utilization and demonstrate that metabolic flux is dynamically gated across the day. Our findings establish a framework for understanding how circadian misalignment contributes to metabolic dysfunction and disease.

NSAIDs are the most widely used medications for the management of chronic pain; however, they are associated with numerous gastrointestinal (GI) adverse events. Although many mechanisms have been suggested, NSAID-induced enteropathy is thought to be primarily due to inhibition of both COX-1 and-2, which results in suppression of prostaglandin synthesis. Yet surprisingly, we found that concomitant postnatal deletion of Cox-1 and-2 over 10 months failed to cause intestinal injury in mice unless they were treated with naproxen or its structural analog, phenylpropionic acid, which is not a COX inhibitor. Cox double-knockout mice exhibited a distinct gut microbiome composition, and cohousing them with controls rescued their dysbiosis and delayed the onset of NSAID-induced GI bleeding. In both the UK Biobank and All of Us human cohorts, coadministration of antibiotics with NSAIDs was associated with an increased frequency of GI bleeding. These results showed that prostaglandin suppression played a trivial role in NSAID-induced enteropathy. However, Cox deletion caused dysbiosis of the gut microbiome, which amplified the enteropathic response to NSAIDs.

Abstract Introduction Sleep enhances procedural and declarative memory consolidation in healthy adults, but individuals with insomnia exhibit deficits in sleep-dependent memory processes. The mechanisms underlying these deficits are unclear. While subjective complaints define insomnia, some individuals also experience objectively short sleep (< 6 h/night). Here, we aimed to disentangle the effects of subjective insomnia and objective short sleep on overnight memory consolidation. Methods Thirty-eight participants were recruited into four groups: insomnia with short sleep (n=5), insomnia with normal sleep (n=13), short sleepers without insomnia (n=4), and good sleepers (n=16). Group classification was based on clinical interview and at-home actigraphy. Participants completed a four-day inpatient protocol that included training and immediate testing on the procedural Motor Sequence Task (MST) and declarative Word Pairs Task (WPT). Delayed retesting occurred after either a night of habitual-length sleep (monitored via polysomnography [PSG]) or an equivalent period of wakefulness. Linear mixed-effects models examined the interaction of condition (Sleep vs Wake), insomnia status, and habitual short sleep on percent change in performance from testing to retesting. Follow-up analyses evaluated potential moderation by PSG parameters. Results The three-way interaction among insomnia, short sleep, and sleep/wake condition did not predict changes in MST or WPT performance. However, a significant interaction between insomnia status and condition emerged for the MST (p=0.02), such that only participants without insomnia benefitted from sleep (Sleep–Wake=8.36% change; p=0.04) whereas participants with insomnia did not (Sleep–Wake=-5.20% change; p=0.22). On the WPT, no interaction was observed, but a significant main effect of condition indicated that all participants benefited from sleep relative to wake (Sleep–Wake=6.32% change, p=0.03). No PSG parameters, including total sleep time, significantly moderated these effects. Conclusion Here, only individuals without subjective sleep complaints benefited from a sleep-dependent boost in procedural memory, but all participants benefited in the declarative memory domain. Habitual sleep duration did not significantly influence outcomes, although there was a limited sample size for short sleepers. Future analyses will explore whether particular EEG features, such as cortical hyperarousal and spindles, mediate relationships between insomnia and performance. Support (if any) This work was supported by the NIH (R01NR018836).

Sleep disruption is an early and prevalent feature of neurodegenerative disease, commonly attributed to neuronal circuit dysfunction or cell loss. However, sleep is tightly coupled to metabolic state, raising the possibility that systemic metabolic abnormalities contribute to disease-associated sleep phenotypes. Using Drosophila models of TDP-43 proteinopathy, we investigated whether peripheral metabolic dysfunction plays a causal role in sleep disruption. We show that TDP-43 expression induces a chronic, starvation-like metabolic state characterized by depletion of peripheral carbohydrate and lipid stores despite normal feeding. Pharmacological restoration of sleep fails to correct these metabolic defects, whereas improving peripheral metabolic state through dietary or genetic interventions robustly rescues sleep. An RNAi-based modifier screen identifies Salt-inducible kinase 3 (SIK3) as a potent suppressor of both sleep loss and starvation sensitivity. Transcriptomic and spatial metabolomic analyses reveal that SIK3 selectively remodels a peripheral metabolic program centered on the pentose phosphate pathway and redox-associated metabolites without globally restoring energy stores. Together, these findings identify systemic metabolic dysfunction as a key driver of sleep disruption in TDP-43 proteinopathy and highlight peripheral metabolism as a potential therapeutic entry point for sleep dysfunction in neurodegenerative disease.

Abstract Activating Transcription Factor 4 (ATF4) is a central mediator of the Integrated Stress Response (ISR), a conserved adaptive pathway activated under conditions of nutrient deprivation, hypoxia, and oxidative stress. In pancreatic ductal adenocarcinoma (PDAC), this pathway supports tumor survival and contributes to the establishment of an immunosuppressive tumor microenvironment (TME). Here, we demonstrate that global ATF4 deletion in the host - affecting the non-tumor, non-CAR T compartments - in combination with mesothelin-CAR T cell therapy, significantly delays tumor progression in syngeneic PDAC models. ATF4 loss in the host was associated with reduced numbers of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), alongside enhanced cytotoxicity and reduced exhaustion in both meso-CAR T and endogenous CD8+ T cells. Spatial metabolomic profiling revealed that ATF4-deficient tumors exhibit focal oxidative stress and depletion of key amino acids, including leucine, suggesting impaired metabolic adaptability that may hinder Treg and MDSC maintenance. Preliminary in vitro differentiation assays further showed that ATF4 loss or pharmacologic ISR inhibition with ISRIB impaired Treg polarization under amino acid-limiting conditions, indicating a metabolic link between ATF4 activity and Treg stability. Importantly, in vivo ISRIB treatment recapitulated the host ATF4-knock out effects, leading to slower tumor growth and improved CAR T cell responses. Finally, combinatorial experiments with anti-PD-1 and anti-CTLA-4 therapy revealed enhanced tumor control in ATF4-KO mice, suggesting that targeting the ISR can sensitize PDAC to immune checkpoint blockade. Collectively, these findings identify ATF4 as a key regulator of PDAC immune evasion and establish ISR inhibition as a promising strategy to enhance both CAR T-cell and checkpoint immunotherapy efficacy in pancreatic cancer. Citation Format: Nektarios Kostopoulos, Rohan Ganesh, Tej Patel, Frank Chinga, Dan Boehmler, Arjun Sengupta, Aalim Weljie, Costa Koumeis, . Genetic and pharmacologic targeting of host ATF4 sensitizes pancreatic ductal adenocarcinoma to CAR T and immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1566.

Sleep disruption is an early and prevalent feature of neurodegenerative disease, commonly attributed to neuronal circuit dysfunction or cell loss. However, sleep is tightly coupled to metabolic state, raising the possibility that systemic metabolic abnormalities contribute to disease-associated sleep phenotypes. Using Drosophila models of TDP-43 proteinopathy, we investigated whether peripheral metabolic dysfunction plays a causal role in sleep disruption. We show that TDP-43 expression induces a chronic, starvation-like metabolic state characterized by depletion of peripheral carbohydrate stores despite normal feeding. Restoration of sleep fails to correct these metabolic defects, whereas improving peripheral metabolic state robustly rescues sleep. A modifier screen of ~650 RNAi lines identified Salt-inducible kinase 3 (SIK3) as a potent suppressor of both sleep loss and starvation sensitivity. Transcriptomic and spatial metabolomic analyses reveal that SIK3 selectively remodels a peripheral metabolic program centered on the pentose phosphate pathway and redox-associated metabolites without globally restoring energy stores. Together, these findings identify systemic metabolic dysfunction as a key driver of sleep disruption in TDP-43 proteinopathy and highlight peripheral metabolism as a potential therapeutic entry point for sleep dysfunction in neurodegenerative disease.

Abstract Introduction Research on the role of circadian rhythms in Insomnia Disorder are mixed. Some studies in small samples have found alterations in melatonin patterns, with reduced levels at night compared to good sleepers, while other studies have failed to confirm these patterns. It can be difficult to draw conclusions because most studies either used melatonin sampling only at night or did not control for lighting levels. The current study examined group differences in melatonin secretion over two days in controlled settings. Methods Forty subjects (20 good sleepers, 20 with insomnia) were recruited to spend 4 nights in a laboratory setting as part of a larger study. Subjects (50% male, 50% female, mean age (SD)=33.5 (7.2)) were kept for 48 hours in light settings below 100 lux and had an IV placed for blood sampling every 2 hours over a 48-hour period. Plasma samples were analyzed with a melatonin radioimmunassay. Melatonin profiles were modeled with a cosinor analysis to compute mesor, acrophase, and amplitude. Results Melatonin parameters did not differ significantly between good sleeper and those with insomnia. Although the insomnia group had a higher mesor value (M=108.32, SD=103.95) than good sleepers (M=77.07, SD=59.02), this difference was not statistically significant (p=0.33). Amplitude between good sleepers (M=294.66, SD=742.40) and those with insomnia (M=107.69, SD=81.12) was also not significant (p=0.34). Finally, acrophase values were nearly identical between participants with insomnia (M=4.15, SD=3.47) and healthy subjects (M=4.28, SD=3.07) (p=0.92). Conclusion The current findings suggest that Insomnia Disorder may not be strongly associated with abnormalities in circadian rhythms of melatonin at the group level. However, the variability observed in melatonin parameters, particularly mesor and amplitude, suggests individual variability that may hide clinically relevant subpopulations. These findings are consistent with clinical trial evidence showing that exogenous melatonin administration is generally not effective for most individuals with insomnia, though some may benefit. Support (if any) NIH/NINR 5R01NR018836

The molecular clock regulates diverse aspects of human biology. As people age, diurnal rhythms deteriorate, most evidently in the daytime napping and nighttime waking of older individuals. To understand how temporal deconsolidation of oscillatory networks could contribute to age-related disease expression, we studied the chronobiome at unprecedented depth in young and old apparently healthy individuals. Transomic integration segregated age groups and identified candidate mechanisms by which oscillatory function might contribute to age dependent distinctions. In an orthogonal approach, we validated as true cyclers many proteins identified in the UK Biobank as predictors of health and disease outcomes. Here, age-specific alterations in the cycling proteome across disease phenotypes is consistent with our hypothesis that deconsolidated circadian programs associate with increased susceptibility to age-related disease.

Cerebral Malaria (CM) is a life-threatening disease caused by Plasmodium falciparum. There are currently no reliable tools to predict the progression of cerebral syndromes in humans. Cerebral malaria results in mortality for approximately 1–2% of affected patients, with diagnosis generally established following the onset of neurological manifestations—such as coma, paralysis, or seizures—which may also be indicative of alternative medical conditions. More research is warranted to detect early biomolecular fingerprints of CM in biofluids. In this study, we have investigated the disease's early molecular markers in mice's urine. Our analysis has identified a significant decrease in methylmalonate in urine for mice with CM, a finding that not only provides a potential early detection tool for CM but also opens new avenues for research and intervention strategies.

Non-steroidal anti-inflammatory drugs are popular choices for the mitigation of pain and inflammation; however, they are accompanied by adverse effects in the gastrointestinal and cardiovascular systems. Identifying biomarkers and mechanisms for early gastrointestinal or cardiovascular disease detection is desirable. Here we compare the effects of placebo, naproxen, a traditional NSAID, and celecoxib, a cyclooxygenase 2 inhibitor and find a decrease in tryptophan and kynurenine levels in the plasma of healthy volunteers who receive naproxen. We further validate this result in mice. Depression of tryptophan is independent of inhibition of either cyclooxygenase but rather, is due to the displacement of bound tryptophan by naproxen. Supplementation of tryptophan in naproxen-treated mice rescues fecal blood loss and inflammatory gene expression driven by IL-1β in the heart. Furthermore, tryptophan also rescues changes in genes that are reflective of inflammation and tissue damage in the gut.

Introduction: Ferroptosis, an iron mediated regulated form of cell death has been implicated in myocardial ischemia-reperfusion (IR) injury among other cardiac conditions. Cellular lipid metabolism and lipid oxidation are key processes in ferroptosis. Circadian clocks, transcriptional feedback loops present in all tissues, regulate diurnal variations in various cardiac physiologic processes including the response to IR injury. The degree to which the clock machinery regulates ferroptosis is unknown. We hypothesize that ferroptosis pathways are induced differently in response to ischemia reperfusion injury at different times of day. Methods: We housed wild-type C57BL6/J mice at a regular 12:12 light dark cycle. We performed a closed chest ischemia reperfusion (IR) procedure to the left anterior descending coronary artery either 2 hours post lights on (Zeitgeber Time 2) or 2 hours post lights off (ZT 14). We sacrificed the mice 24 hours post reperfusion and collected hearts for molecular clock and ferroptosis gene expression quantification by RT-qPCR and to measure a large panel of oxidized lipids and metabolites of oxidation pathways. Because the surgical mice were sacrificed at opposing times of day, we accounted for normal day to night variations by reporting all measurements as the number of standard deviations away from the measurement mean of non-surgical mice sacrificed at the same time of day. Results: The expression of the anti-ferroptosis molecular clock gene Bmal1 was decreased beyond its normal diurnal variations in the mice with the IR procedure at ZT2 compared to those at ZT14. Furthermore, the expression levels of ferroptosis genes Alox15 and Acsl4 were higher in the ZT2 group compared to ZT14 indicating higher induction of ferroptosis in the ZT2 group beyond that of normal diurnal variation. From our metabolomics panel, we find a higher ratio of NADP+ to NADPH in the ZT 2 group indicating a higher redox burden. Also, acyl carnitines of differing chain lengths showed a consistent decrease in the ZT 2 group. Conclusions: In response to ischemia reperfusion injury, ferroptosis is induced in a diurnal manner reflective of alterations in the core clock gene Bmal1, lipid metabolism and oxidation pathways.

Resistance to chemotherapy is an important challenge in the clinical management of triple-negative breast cancer (TNBC). Utilization of the amino acid glutamine as a key nutrient is a metabolic signature of TNBC featuring high glutaminase (GLS) activity and a large pool of cellular glutamate, which mediates intracellular enrichment of cystine via xCT (SLC7A11) antiporter activity. To overcome chemo-resistant TNBC, we identified a strategy of dual metabolic inhibition of GLS and xCT to sensitize resistant TNBC cells to chemotherapy. We successfully tested this strategy in a human TNBC line and its chemoresistant variant in vitro and their xenograft models in vivo. Key findings of our study include: 1. Dual metabolic inhibition induced pronounced reductions of cellular glutathione accompanying significant increases of cellular superoxide level in both parent and resistant TNBC cells. While GLS and xCT inhibition did not directly kill cells via apoptosis, they potentiated doxorubicin (DOX) and cisplatin (CIS) to induce remarkably higher levels of apoptosis than DOX or CIS alone. 2. Although the resistant TNBC cells exhibited higher capacity to mitigate oxidative stress than the parent cells, their resistance was overcome by dual metabolic inhibition combined with DOX or CIS. 3. In vivo efficacy and safety of the triple combination (GLS and xCT inhibition plus DOX or CIS) were demonstrated in both chemo sensitive and resistant TNBC tumors in mice. In conclusion, GLS and xCT inhibition resulted in unmitigated oxidative stress due to depletion of glutathione, representing a promising strategy to overcome chemoresistance in glutamine-dependent TNBC.

Precise temporal regulation of metabolism by sleep and circadian rhythms is essential for dynamic energy homeostasis, yet the link between systemic metabolism and respiratory demands remains poorly defined. We combined high-resolution respirometry with LC-MS-based metabolomics to characterize respiratory dynamics and metabolic states in Drosophila melanogaster to uncover genotype-specific impacts of sleep and circadian disruption. Wild-type flies under light-dark cycles (WT-LD) showed rhythmic respiratory patterns reflective of anticipatory coordination of mitochondrial energy metabolism, amino acid turnover, and redox cycling. In contrast, short-sleep mutants (fmn, sss) exhibited elevated metabolic rates and reactive shifts of fuel preferences toward lipid and amino acid catabolism and displayed signs of mitochondrial stress. Circadian-clock disrupted flies (per 01 , WT-DD) showed reactive and widespread metabolic dysregulation and impaired redox homeostasis. These findings demonstrate that both sleep and circadian systems are essential for aligning metabolic substrate selection with energy demands, offering mechanistic insights into how disruptions in behavioral states compromise metabolic health.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used medications for the management of chronic pain; however, they are associated with numerous gastrointestinal (GI) adverse events. Although many mechanisms have been suggested, NSAID-induced enteropathy has been thought to be primarily due to inhibition of both cyclooxygenases (COX) -1 and -2, which results in suppression of prostaglandin synthesis. Yet surprisingly, we found that concomitant postnatal deletion of Cox-1 and -2 over 10 months failed to cause intestinal injury in mice unless they were treated with naproxen or its structural analog, phenylpropionic acid, which is not a COX inhibitor. Cox double knockout mice exhibit a distinct gut microbiome composition and cohousing them with controls rescues their dysbiosis and delays the onset of NSAID-induced GI bleeding. In both the UK Biobank and All of Us human cohorts, coadministration of antibiotics with NSAIDs is associated with an increased frequency of GI bleeding. These results show that prostaglandin suppression plays a trivial role in NSAID-induced enteropathy. However, Cox deletion causes dysbiosis of the gut microbiome that amplifies the enteropathic response to NSAIDs.

Mass Spectrometry Imaging (MSI) is an emergent tool for analyzing spatial molecular distributions, yet data complexity often hinders effective analysis. MSI.EAGLE, an open-source R-Shiny application, makes analysis accessible for non-specialists by integrating advanced tools in a user-friendly interface. The workflow leverages tools from the Cardinal MSI package, with enhanced phenotyping, segmentation, statistical analysis and visualization. The application addresses a gap in spatial biology research by empowering a broader scientific community.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1–derived thromboxane A 2 . Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1 flox/ flox mice to generate platelet Cox-1 −/− mice on normolipidemic and hyperlipidemic (Ldlr −/− ) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1 −/− /Ldlr −/− and Gp1ba-ΔCre Cox-1 −/− /Ldlr −/− mice. In a mouse model of tail injury, Pf4-ΔCre–mediated and Gp1ba-ΔCre–mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI 2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE 2 and PGD 2 , activation of the inflammasome, elevated plasma levels of IL-1β, reduced plasma levels of HDL-C, and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1–dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Sickness sleep and rebound following sleep deprivation share humoral signals including the rise of cytokines, in particular interleukins. Nevertheless, they represent unique physiological states with unique brain firing patterns and involvement of specific circuitry. Here, we performed untargeted metabolomics of mouse cortex and hippocampus to uncover changes with sickness and rebound sleep as compared with normal daily sleep. We found that the three settings are biochemically unique with larger differences in the cortex than in the hippocampus. Both sickness and rebound sleep shared an increase in tryptophan. Surprisingly, these two sleep conditions showed opposite modulation of the methionine-homocysteine cycle and differences in terms of the energetic signature, with sickness impinging on glycolysis intermediates whilst rebound increased the triphosphorylated form of nucleotides. These findings indicate that rebound following sleep deprivation stimulates an energy rich setting in the brain that is devoid during sickness sleep.

Rhythmicity is a cornerstone of behavioral and biological processes, especially metabolism, yet the mechanisms behind metabolite cycling remain elusive. This study uncovers a robust oscillation in key metabolite pathways downstream of glucose in humans. A purpose-built 13C6-glucose isotope tracing platform was used to sample Drosophila every 4h and probe these pathways, revealing a striking peak in biosynthesis shortly after lights-on in wild-type flies. A hyperactive mutant (fumin) demonstrates increased Krebs cycle labelling and dawn-specific glycolysis labelling. Surprisingly, neither underlying feeding rhythms nor the presence of food availability explain the rhythmicity of glucose processing across genotypes, suggesting a robust internal mechanism for metabolic control of glucose processing. These results align with clinical data highlighting detrimental effects of mistimed energy intake. Our approach offers a unique insight into the dynamic range of daily metabolic processing and provides a mechanistic foundation for exploring circadian metabolic homeostasis in disease contexts.