SUMMARY Circadian clocks and metabolism are inextricably intertwined, where central and hepatic circadian clocks coordinate metabolic events in response to light-dark and sleep-wake cycles. We reveal an additional key element involved in maintaining host circadian rhythms, the gut microbiome. Despite persistence of light-dark signals, germ-free mice fed low or high fat diets exhibit markedly impaired central and hepatic circadian clock gene expression and do not gain weight compared to conventionally-raised counterparts. Examination of gut microbiota in conventionally-raised mice showed differential diurnal variation in microbial structure and function dependent upon dietary composition. Additionally, specific microbial metabolites induced under low or high fat feeding, particularly short chain fatty acids, but not hydrogen sulfide, directly modulate circadian clock gene expression within hepatocytes. These results underscore the ability of microbially-derived metabolites to regulate or modify central and hepatic circadian rhythm and host metabolic function, the latter following intake of a Westernized diet.

The gut microbiota play important roles in lipid metabolism and absorption. However, the contribution of the small bowel microbiota of mammals to these diet-microbe interactions remains unclear. We determine that germ-free (GF) mice are resistant to diet-induced obesity and malabsorb fat with specifically impaired lipid digestion and absorption within the small intestine. Small bowel microbes are essential for host adaptation to dietary lipid changes by regulating gut epithelial processes involved in their digestion and absorption. In addition, GF mice conventionalized with high-fat diet-induced jejunal microbiota exhibit increased lipid absorption even when fed a low-fat diet. Conditioned media from specific bacterial strains directly upregulate lipid absorption genes in murine proximal small intestinal epithelial organoids. These findings indicate that proximal gut microbiota play key roles in host adaptability to dietary lipid variations through mechanisms involving both the digestive and absorptive phases and that these functions may contribute to conditions of over- and undernutrition.

We recently reported that differentiation of CD8 1 T cells from the naïve to the effector state involves the upregulation of glucose-dependent metabolism. Glucose deprivation or inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) selectively inhibited production of IFN-g but not of IL-2. To determine a more global role of glucose metabolism on effector T-cell function, we performed gene array analysis on CD8 1 effector T cells stimulated in the presence or absence of 2-DG. We observed that expression of only 10% of genes induced by TCR/CD28 signaling was inhibited by 2-DG. Among these were genes for key cytokines, cell cycle molecules, and cytotoxic granule proteins. Consistent with these results, production of IFN-g and GM-CSF, cell cycle progression, upregulation of cyclin D2 protein, cytolytic activity, and upregulation of granzyme B protein and also conjugate formation were exquisitely glucose-dependent. In contrast to glucose, oxygen was little utilized by CD8 1 effector T cells, and relative oxygen deprivation did not inhibit these CTL functional properties. Our results indicate a particularly critical role for glucose in regulating specific effector functions of CD8 1 T cells and have implications for the maintenance of the effector phase of cellular immune responses in target tissue microenvironments such as a solid tumor.

Differentiation of CD8+ T cells from the naive to the effector state is accompanied by changes in basal gene expression profiles that parallel the acquisition of effector functions. Among these are metabolism genes, and we now show that 2C TCR transgenic effector CD8+ T cells express higher levels of glycolytic enzymes and display greater glucose uptake, a higher glycolytic rate, and increased lactate production compared with naive cells. To determine whether glucose was required for effector T cell functions, we regulated glucose availability in vitro. Glucose deprivation strongly inhibited IFN-γ gene expression, whereas IL-2 production was little affected. Inhibition correlated with diminished phosphorylation of p70S6 kinase and eIF4E binding protein 1 and a requirement for de novo protein synthesis, whereas other signaling pathways known to regulate IFN-γ expression were unaffected. Together, our data reveal that optimal induction of IFN-γ transcription is a glucose-dependent process, indicate that there are undefined factors that influence IFN-γ expression, and have implications for regulation of the effector phase of CD8+ T cell responses in tissue microenvironments.

SUMMARY Transcriptional regulation of circadian rhythms is essential for lipid metabolic homeostasis, disruptions of which can lead to metabolic diseases. Whether N6-methyladenosine (m6A) mRNA methylation impacts circadian regulation of lipid metabolism is unclear. Here, we show m6A mRNA methylation oscillations in murine liver depend upon a functional circadian clock. Hepatic deletion of Bmal1 increases m6A mRNA methylation, particularly of PPaRα. Inhibition of m6A methylation via knockdown of m6A methyltransferase METTL3 decreases PPaRα m6A abundance and increases PPaRα mRNA lifetime and expression, reducing lipid accumulation in cells in vitro. Mechanistically, YTHDF2 binds to PPaRα to mediate its mRNA stability to regulate lipid metabolism. Induction of reactive oxygen species both in vitro and in vivo increases PPaRα transcript m6A levels, revealing a possible mechanism for circadian disruption on m6A mRNA methylation. These data show that m6A RNA methylation is important for circadian regulation of downstream genes and lipid metabolism, impacting metabolic outcomes.