Transitions in chromatin conformation shaped by fatty acids and the circadian clock underlie hepatic transcriptional reorganization in obese mice.

The circadian clock system coordinates metabolic, physiological, and behavioral functions across a 24-h cycle, crucial for adapting to environmental changes. Disruptions in circadian rhythms contribute to major metabolic pathologies like obesity and Type 2 diabetes. Understanding the regulatory mechanisms governing circadian control is vital for identifying therapeutic targets. It is well characterized that chromatin remodeling and 3D structure at genome regulatory elements contributes to circadian transcriptional cycles; yet the impact of rhythmic chromatin topology in metabolic disease is largely unexplored. In this study, we explore how the spatial configuration of the genome adapts to diet, rewiring circadian transcription and contributing to dysfunctional metabolism. We describe daily fluctuations in chromatin contacts between distal regulatory elements of metabolic control genes in livers from lean and obese mice and identify specific lipid-responsive regions recruiting the clock molecular machinery. Interestingly, under high-fat feeding, a distinct interactome for the clock-controlled gene Dbp strategically promotes the expression of distal metabolic genes including Fgf21. Alongside, new chromatin loops between regulatory elements from genes involved in lipid metabolism control contribute to their transcriptional activation. These enhancers are responsive to lipids through CEBPß, counteracting the circadian repressor REVERBa. Our findings highlight the intricate coupling of circadian gene expression to a dynamic nuclear environment under high-fat feeding, supporting a temporally regulated program of gene expression and transcriptional adaptation to diet.

Time-of-day defines NAD+ efficacy to treat diet-induced metabolic disease by synchronizing the hepatic clock in mice.

The circadian clock is an endogenous time-tracking system that anticipates daily environmental changes. Misalignment of the clock can cause obesity, which is accompanied by reduced levels of the clock-controlled, rhythmic metabolite NAD+. Increasing NAD+ is becoming a therapy for metabolic dysfunction; however, the impact of daily NAD+ fluctuations remains unknown. Here, we demonstrate that time-of-day determines the efficacy of NAD+ treatment for diet-induced metabolic disease in mice. Increasing NAD+ prior to the active phase in obese male mice ameliorated metabolic markers including body weight, glucose and insulin tolerance, hepatic inflammation and nutrient sensing pathways. However, raising NAD+ immediately before the rest phase selectively compromised these responses. Remarkably, timed NAD+ adjusted circadian oscillations of the liver clock until completely inverting its oscillatory phase when increased just before the rest period, resulting in misaligned molecular and behavioral rhythms in male and female mice. Our findings unveil the time-of-day dependence of NAD+-based therapies and support a chronobiology-based approach.

Astrocytic circadian clock control of energy expenditure by transcriptional stress responses in the ventromedial hypothalamus.

Hypothalamic circuits compute systemic information to control metabolism. Astrocytes residing within the hypothalamus directly sense nutrients and hormones, integrating metabolic information, and modulating neuronal responses. Nevertheless, the role of the astrocytic circadian clock on the control of energy balance remains unclear. We used mice with a targeted ablation of the core-clock gene Bmal1 within Gfap-expressing astrocytes to gain insight on the role played by this transcription factor in astrocytes. While this mutation does not substantially affect the phenotype in mice fed normo-caloric diet, under high-fat diet we unmasked a thermogenic phenotype consisting of increased energy expenditure, and catabolism in brown adipose and overall metabolic improvement consisting of better glycemia control, and body composition. Transcriptomic analysis in the ventromedial hypothalamus revealed an enhanced response to moderate cellular stress, including ER-stress response, unfolded protein response and autophagy. We identified Xbp1 and Atf1 as two key transcription factors enhancing cellular stress responses. Therefore, we unveiled a previously unknown role of the astrocytic circadian clock modulating energy balance through the regulation of cellular stress responses within the VMH.

Coordinated metabolic transitions and gene expression by NAD+ during adipogenesis.

Adipocytes are the main cell type in adipose tissue, which is a critical regulator of metabolism, highly specialized in storing energy as fat. Adipocytes differentiate from multipotent mesenchymal stromal cells (hMSCs) through adipogenesis, a tightly controlled differentiation process involving close interplay between metabolic transitions and sequential programs of gene expression. However, the specific gears driving this interplay remain largely obscure. Additionally, the metabolite nicotinamide adenine dinucleotide (NAD+) is becoming increasingly recognized as a regulator of lipid metabolism, and a promising therapeutic target for dyslipidemia and obesity. Here, we explored how NAD+ bioavailability controls adipogenic differentiation from hMSC. We found a previously unappreciated repressive role for NAD+ on adipocyte commitment, while a functional NAD+-dependent deacetylase SIRT1 appeared crucial for terminal differentiation of pre-adipocytes. Repressing NAD+ biosynthesis during adipogenesis promoted the adipogenic transcriptional program, while two-photon microscopy and extracellular flux analyses suggest that SIRT1 activity mostly relies on the metabolic switch. Interestingly, SIRT1 controls subcellular compartmentalization of redox metabolism during adipogenesis.

Circadian Regulation of Immunity Through Epigenetic Mechanisms.

The circadian clock orchestrates daily rhythms in many physiological, behavioral and molecular processes, providing means to anticipate, and adapt to environmental changes. A specific role of the circadian clock is to coordinate functions of the immune system both at steady-state and in response to infectious threats. Hence, time-of-day dependent variables are found in the physiology of immune cells, host-parasite interactions, inflammatory processes, or adaptive immune responses. Interestingly, the molecular clock coordinates transcriptional-translational feedback loops which orchestrate daily oscillations in expression of many genes involved in cellular functions. This clock function is assisted by tightly controlled transitions in the chromatin fiber involving epigenetic mechanisms which determine how a when transcriptional oscillations occur. Immune cells are no exception, as they also present a functional clock dictating transcriptional rhythms. Hereby, the molecular clock and the chromatin regulators controlling rhythmicity represent a unique scaffold mediating the crosstalk between the circadian and the immune systems. Certain epigenetic regulators are shared between both systems and uncovering them and characterizing their dynamics can provide clues to design effective chronotherapeutic strategies for modulation of the immune system.

Two novel asparaginyl endopeptidase-like cysteine proteinases from the protist Trichomonas vaginalis: their evolutionary relationship within the clan CD cysteine proteinases.

Cysteine proteinases (CPs) are important virulence factors of the protozoan parasite Trichomonas vaginalis. A total of six genes coding for cathepsin L-like CPs belonging to clan CA have been identified in T. vaginalis. At least 23 distinct spots with proteolytic activity have been detected by two-dimensional (2-D) substrate gel electrophoresis from in vitro grown parasites; however, only few of them have been characterized. In this work, we detected six spots with proteolytic activity and molecular weights between 25 and 35 kDa. The six proteinases correspond to two distinct CP families: the papain-like family, represented by four spots with pIs between 4.5 and 5.5; and the legumain-like family represented by two spots with pI 6.3 and 6.5. Next, we obtained two cDNAs encoding for legumain-like CPs from T. vaginalis, which were named Tvlegu-1 and Tvlegu-2. The size of these cDNA clones were 1225 and 1364 bp, which encoded for 388 and 415 amino acids, respectively. Their putative translation products have molecular masses of 42.8 and 47.2 kDa, corresponding to inactive legumain-like CP precursors. The two sequences share approximately 40% identity at the amino acid level. These protein products can be classified within a branch of the legumain-like family in clan CD cysteine proteinases due to their sensitivity to specific proteinases inhibitors, their DNA sequences, and phylogenetic reconstruction. However, they do not correspond either to the typical asparaginyl endopeptidase or the glycosylphosphatidylinositol (GPI): protein transamidase subfamilies. These results suggest that the TVLEGU-1 and TVLEGU-2 peptidases are likely to be part of a new subfamily within the legumain-like family of clan CD cysteine proteinases. Furthermore, they could be one of the missing links between prokaryotic and eukaryotic CPs in clan CD enzymes.