Circadian rhythm disruption profoundly alters host physiology and increases susceptibility to metabolic and inflammatory conditions (4). Given the intimate role of gut microbiota and the eCBome in regulating multiple aspects of metabolic health (47, 61) and responding to circadian cycling (26, 50), we went on to determine how circadian rhythm disruption using constant light modulates eCBome lipid levels and gut bacteria. We selected ZT11 and CT11 for organ collection because it represents a transition point into the dark cycle and active phase, making it ideal for studying metabolic and circadian-regulated processes (27). Our findings reveal clear sex-dependent adaptive strategies to constant light (LL) with respect to changes induced in bioactive lipid levels in different organs and fecal bacteria composition.

LL exposure altered the gut microbiota in a sex-dependent manner. In females, taxa such as Rikenellaceae, Butyricicoccaceae, Alistipes, Eisenbergiella, and Enterorhabdus were enriched, while Erysipelotrichaceae, Turicibacter, and Ruminococcaceae UCG-005 were depleted. Males showed fewer compositional shifts, with increases in Tuzzerella and Oscillospirales-UCG-010 and reductions in Ruminococcaceae. Several of these taxa have been linked to circadian disruption or rhythmicity in prior studies. For example, in constant darkness (DD), Alistipes (62) significantly changes in the opposite direction to our protocol (constant light), whereas Rikenellaceae (63) increased in the same direction. These differences likely reflect the distinct circadian stressors of LL versus DD (loss of light–dark cycles vs. loss of photic cues), highlighting their sensitivity to circadian misalignment.

Indeed intestinal epithelium disruption of Bmal1 leading to disruption of the intestinal clock disrupts Alistipes rhythmicity (64), and constant light exposure causes the opposite effect on its levels compared to what we report here (64).Thus our data suggests that this genus may be particularly sensitive to light exposure, though the potential effects of this would be dependent on the species affected, as different Alistipes spp. have been found to associate with positive or negative physiological effects (65). The consistent reduction of Turicibacter, a genus positively associated with metabolic health, potentially by modifying bile acids and thus affecting lipid metabolism (66), further suggests that LL disrupts microbial taxa normally linked to rhythmicity and beneficial functions.

Despite pronounced taxonomic alterations in females, SCFA production increased only in males, with higher levels of isobutyric, butyric, isovaleric, and valeric acids. These shifts coincided with reductions in the Ruminococcaceae family and enrichment of Oscillospirales-UCG-010 and Tuzzerella at the genus level. The Oscillospirales-UCG-010 correlate with SCFA levels (67). This enrichment is consistent with the elevated fecal SCFA levels observed in males, suggesting a functional link between microbial composition and metabolite output. Tuzzerella, although not a major SCFA producer, has been reported to increase in models of gut inflammation (68), where it modulates immune responses by regulating T cell infiltration (69). Its enrichment under LL may therefore reflect proliferation during inflammatory stress. In parallel, SCFAs such as acetate, propionate, and butyrate can enter the liver and directly modulate circadian clock gene expression (51). SCFAs also exert local benefits, including reducing systemic inflammation, improving hepatic lipid metabolism, and ameliorating mucosal injury in the small intestine (70). Given established associations between SCFAs and sleep duration (71), their elevation in males may indicate a behavioral or metabolic protective adaptation to LL-induced circadian rhythm disruption. These elevated levels of SCFAs might potentially have affected the levels of oxylipins within the distal intestinal tract, which in fact increased in the duodenum and jejunum of male LL mice, and decreased in females. Indeed, in comparison to the duodenum, the lower intestinal regions showed also significant increases in long-chain PUFAs, including DHA and its LOX-derived oxylipins, accompanied by suppression of NAEs and prostaglandins, emphasizing the distal gut as a hotspot of circadian stress responses.

In females, although the microbial community composition changed markedly, SCFA levels did not. Instead, intestinal lipid remodeling reflected widespread depletion of protective mediators. The proximal small intestine showed downregulation of fatty acids (LA, OA), MAGs (2-SDG), and prostaglandins (PGE₂). NAEs were largely unchanged in the duodenum and jejunum but markedly suppressed in the ileum (LEA, PEA). While DHA was increased in the ileum where, unlike males, its oxidized metabolites were not. These signatures align with prior reports linking circadian disruption to intestinal inflammation (72) and heightened metabolic disease susceptibility (73, 74). Such sex-specific differences may reflect greater disruption of intestinal clock gene expression and metabolic adaptation in females (75) and their propensity of female mice to lose weight under protocols of light-cycle disruption while males gain weight (76).

Under LL, females showed suppression of lipid mediators in the proximal intestine but activation in the distal gut, muscle, and reproductive organs (e.g., DPA n-3, 2-AG, 15-HpEDE, PGD[), indicating a redistribution of lipid signaling from the gut toward peripheral tissues. Females engaged NAE-centered eCBome signaling (e.g., AEA, OEA, PEA) in the brain and liver, accompanied by systemic inflammatory cytokine responses. In contrast, males exhibited activation of oxylipin and 2-MAG pathways in the intestine and liver (e.g., 5-HETE, 5-KETE, 10,17-DiHDHA, 14-/ 7-HDHA, 2-AG) and in skeletal muscle (e.g., 2-AG, 2-LG). In the adipose tissues, particularly inguinal WAT, males showed coordinated suppression of anti-inflammatory oxylipins (e.g., 15-HEPE, 5-/ 15-HETE, 14-/ 7-HDHA, 13-HODE-G, 6-keto PGF[α). Overall, males relied on SCFA- and ω-3-derived resolution pathways, particularly in the colon and liver, whereas females engaged eCBome-driven signaling and immune activation under circadian disruption. The liver exhibited a mixed regulatory profile. In females, ω-3 fatty acids (SDA, EPA) decreased alongside increased 2-MAGs (2-SDG, 2-AG). In males, prostaglandins (PGF[α, PGE[) and NAEs (LEA, DHEA) were suppressed, while also in this sex 2-MAGs (2-SDG, 2-OG) increased. When sexes were combined, reduced prostaglandins and NAEs persisted and 2-MAGs remained elevated. This shift mirrors metabolic liver disease, where reduced ω-3 fatty acids (77) and prostaglandins (78) promote accumulation of 2-MAG-derived diacylglycerol (DAG) via monoacylglycerol acyltransferases (MGATs), subsequently forming triacylglycerols (TAGs) through DGAT2 (79), an enzyme that itself exhibits rhythmic expression (80). This pathway contributes to non-alcoholic fatty liver disease (NAFLD) (81). Interestingly, however, 2-SDG emerged as the most responsive hepatic molecule under LL in both sexes. This 2-MAG is derived from the ω-3 fatty acid, SDA, which is associated with potential anti-inflammatory properties (82). However, the precise biological role of 2-SDG remains to be clarified both metabolically and with respect to its potential involvement in light-cycle disruption effects on liver metabolism.

The brain showed a sex dependent response; in females, the cerebellum displayed robust upregulation of NAEs and prostaglandins which is consistent with neuroprotective response of NAEs (83–86). This response was absent in males. The hypothalamus also showed LOX-derived oxylipins (derived from both FAs and 2-MAGs; e.g., 12-HETE, 15-HETE, 15-HETE-G) proposed to modulate neuroinflammation and neuronal lipid signaling (87), reflecting the sensitivity of this brain area to circadian stress. Pharmacological CB₁ activation reduces the phase-shifting effects of light on the suprachiasmatic nucleus in male but not female mice (88), further supporting tissue- and sex-specific differences in circadian responsiveness. By contrast, the midbrain and right hemisphere exhibited marked downregulation of NAEs, 2-MAGs, oxylipins, and long-chain fatty acids (e.g., DGLA, DHA, DPA), with males showing a more prostaglandin-centered response. Although NAEs are generally considered neuroprotective, multiple studies indicate they can also contribute to pathogenesis and elevate disease risk. Elevated NAE levels have been linked with aging, neuroinflammation, and ischemia (89), can directly trigger cell death (90, 91), and are increased in psychotic patients (92). This duality likely reflects the several molecular targets for each of these mediators, ranging from CB₁ and TRPV1, which may play pro-inflammatory and brain function disrupting effects, to GPR110, PPARs and CB₂, which instead play pro-homeostatic anti-inflammatory actions (37). Circadian disturbances are highly prevalent in schizophrenia and psychosis (93) and with aging, the amplitude of circadian rhythms progressively declines (94). Collectively, these findings suggest that female brains preferentially engage NAE pathways as a protective buffer against circadian rhythm disruption. However, given the dual role of NAEs in both protection and pathology, their alteration under light-cycle disruption may lead to maladaptive neurobiological changes in females, potentially contributing to greater vulnerability to shift-work disorder, stress, and insomnia (95).

LL exposure induced FA accumulation across several skeletal muscles, with little or no effect, however, on eCBome mediators. Sleep restriction and loss of circadian rhythmicity are known to cause atrophy and impaired metabolic plasticity in muscles such as the quadriceps, soleus, and gastrocnemius (96, 97). These muscles are enriched for rhythmic genes involved in fatty acid metabolism, mitochondrial oxidative phosphorylation, and glucose handling (98, 99). Moreover, post-flight biopsies of human soleus have demonstrated susceptibility to lipid accumulation under circadian misalignment (100). Within this context, our data show that LA and OA accumulated in the gastrocnemius, and LA in the soleus, indicating maladaptive lipid storage particularly within glycolytic muscle fibers, consistent with reduced oxidative flexibility and altered energy metabolism.

In contrast to muscles the adipose tissue was less effected by circadian rhythm disruption; in inguinal WAT, females showed increased 2-MAGs, including 2-AG, indicating enhanced cannabinoid receptor CB[activity (101). Adipose CB[signaling is known to impair insulin sensitivity, enhance lipogenesis, and suppress mitochondrial oxidative capacity, ultimately promoting lipid accumulation and endoplasmic reticulum stress (102, 103). These changes may therefore represent an early maladaptive metabolic response to circadian disruption. In contrast, males exhibited coordinated suppression of multiple oxylipins and NAEs, suggesting loss of local anti-inflammatory tone that may predispose to low-grade inflammation (104). In retroperitoneal WAT, increases in prostaglandins (PGD[, PGB[) and 2-MAGs (2-AG, 2-DPG, both of which have been shown to have context-dependent anti- and pro-inflammatory activities (43)) highlighting coordinated eCBome response within a given family of lipids. The decreased levels of DGLA may indicate limited substrate availability for the production of anti-inflammatory prostaglandin synthesis and thus a relative increase in pro-inflammatory ones, potentially exacerbating inflammation (105). Interscapular BAT displayed suppression of bioactive lipids including ω-3 fatty acids, EPEA, and prostaglandins, consistent with reduced lipid signaling capacity that could blunt thermogenic and oxidative functions (106). An increase in 2-MAGs across multiple adipose tissues may contribute to metabolic dysfunction and obesity-related complications, such as insulin resistance and type 2 diabetes, through their role as intermediates in triglyceride synthesis (107). Together, these findings align with the LL disruption of adipose lipolysis and its coordination with skeletal muscle fatty acid uptake to cause a systemic metabolic dysfunction (108).

Reproductive tissues displayed a similar remodeling under constant light with having increased the 2-MAGs, and FA-oxylipins in male and female. In particular, the males showed upregulation of OEA, DPA(n-3), PGE[, multiple oxylipins (4-HDHA, 17-oxo-DHA, 5-KETE-G), and 2-MAGs (2-AG, 2-DPG, 2-EPG). In females, we also observed increased in oxylipins (12-oxo-LTB4, 8,15-DiHETE, 17-HDHA), and 2-MAGs (2-AG, 2-DPG) were elevated while DPA(n-6) was downregulated. Both testes and ovaries have functional molecular clocks; in males, these clocks regulate testosterone production, spermatogenesis, and sperm motility (109), and in females, they coordinate hypothalamic-pituitary-gonadal (HPG) axis output with ovarian steroidogenesis and ovulation timing, partly through rhythmic prostaglandin and NAE signaling (110). Disruption of these rhythms is therefore likely to impair fertility. Clinical observations support this interpretation. Elevated seminal 2-AG has been associated with reduced sperm motility in men (111), while high 2-AG levels are reported in women with endometriosis and infertility (112). Similarly, increased oxylipins and associated oxidative stress can impair gamete function and reproductive outcomes (113), linking circadian disruption, dysregulated lipid signaling, and chronic inflammation to infertility. Supporting this link, clinical studies indicate that misalignment of circadian rhythms such as that caused by shift work or constant light, leads to reduced fertility, irregular cycles, and lower conception rates (114).

Systemic cytokine profiling revealed distinct sex-specific immune signatures under constant light. Females displayed elevated IL-5, IFN-γ, and the neutrophil-recruiting chemokine MIP-2, whereas males showed only IL-2 significant increase. This pattern suggests that circadian disruption amplifies immune responsiveness more strongly in females and is consistent with computational and experimental models indicating that circadian clocks modulate immune activity in a sex-dependent manner (115). IFN-γ levels were reported to be significantly altered in Per2 mutant mice, which lacked a daily rhythm (116), and studies in non-human primates showed that the severity of circadian rhythm disturbances correlates with plasma IFN-γ levels (117). Interestingly, despite the heightened cytokine response, plasma lipid mediators showed a predominantly suppressive profile under constant light. In females, both SDA and the oxylipin 12-HHTrE were reduced, while males exhibited lower 2-AG levels.

The present study has some limitations. The relatively small sample size (n=4 per sex for each treatment) may have limited statistical power. We assessed tissues at a single circadian timepoint, which may not capture dynamic oscillations of microbial and lipid networks. Finally, the sex hormone status in females was not considered and could have contributed to variability in circadian responses.