bims-ciryme 2024-04-14 papers

Methods Mol Biol. 2024 ;2795 43-53

A Luciferase Imaging-Based Assay for Studying Temperature Compensation of the Circadian Clock.

The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.

Keywords: Arabidopsis; Bioluminescence; Circadian oscillator; Low-light imaging; Period analysis; Temperature buffering

DOI: https://doi.org/10.1007/978-1-0716-3814-9_5

Sci Rep. 2024 04 07. 14(1): 8128

Excessive fat expenditure in MCT-induced heart failure rats is associated with BMAL1/REV-ERBα circadian rhythmic loop disruption.

Dufang Ma, Yiwei Qu, Tao Wu, Xue Liu, Lu Cai, Yong Wang.

Fat loss predicts adverse outcomes in advanced heart failure (HF). Disrupted circadian clocks are a primary cause of lipid metabolic issues, but it's unclear if this disruption affects fat expenditure in HF. To address this issue, we investigated the effects of disruption of the BMAL1/REV-ERBα circadian rhythmic loop on adipose tissue metabolism in HF.50 Wistar rats were initially divided into control (n = 10) and model (n = 40) groups. The model rats were induced with HF via monocrotaline (MCT) injections, while the control group received equivalent solvent injections. After establishing the HF model, the model group was further subdivided into four groups: normal rhythm (LD), inverted rhythm (DL), lentivirus vector carrying Bmal1 short hairpin RNA (LV-Bmal1 shRNA), and empty lentivirus vector control (LV-Control shRNA) groups, each with 10 rats. The DL subgroup was exposed to a reversed light-dark cycle of 8 h: 16 h (dark: light), while the rest adhered to normal light-dark conditions (light: dark 12 h: 12 h). Histological analyses were conducted using H&E, Oil Red O, and Picrosirius red stains to examine adipose and liver tissues. Immunohistochemical staining, RT-qPCR, and Western blotting were performed to detect markers of lipolysis, lipogenesis, and beiging of white adipose tissue (WAT), while thermogenesis indicators were detected in brown adipose tissue (BAT). The LD group rats exhibited decreased levels of BMAL1 protein, increased levels of REV-ERBα protein, and disrupted circadian circuits in adipose tissue compared to controls. Additionally, HF rats showed reduced adipose mass and increased ectopic lipid deposition, along with smaller adipocytes containing lower lipid content and fibrotic adipose tissue. In the LD group WAT, expression of ATGL, HSL, PKA, and p-PKA proteins increased, alongside elevated mRNA levels of lipase genes (Hsl, Atgl, Peripilin) and FFA β-oxidation genes (Cpt1, acyl-CoA). Conversely, lipogenic gene expression (Scd1, Fas, Mgat, Dgat2) decreased, while beige adipocyte markers (Cd137, Tbx-1, Ucp-1, Zic-1) and UCP-1 protein expression increased. In BAT, HF rats exhibited elevated levels of PKA, p-PKA, and UCP-1 proteins, along with increased expression of thermogenic genes (Ucp-1, Pparγ, Pgc-1α) and lipid transportation genes (Cd36, Fatp-1, Cpt-1). Plasma NT-proBNP levels were higher in LD rats, accompanied by elevated NE and IL-6 levels in adipose tissue. Remarkably, morphologically, the adipocytes in the DL and LV-Bmal1 shRNA groups showed reduced size and lower lipid content, while lipid deposition in the liver was more pronounced in these groups compared to the LD group. At the gene/protein level, the BMAL1/REV-ERBα circadian loop exhibited severe disruption in LV-Bmal1 shRNA rats compared to LD rats. Additionally, there was increased expression of lipase genes, FFA β oxidation genes, and beige adipocyte markers in WAT, as well as higher expression of thermogenic genes and lipid transportation genes in BAT. Furthermore, plasma NT-proBNP levels and adipose tissue levels of NE and IL-6 were elevated in LV-Bmal1 shRNA rats compared with LD rats. The present study demonstrates that disruption of the BMAL1/REV-ERBα circadian rhythmic loop is associated with fat expenditure in HF. This result suggests that restoring circadian rhythms in adipose tissue may help counteract disorders of adipose metabolism and reduce fat loss in HF.

Keywords: BMAL1/REV-ERBα circadian rhythmic loop; Beiging of white adipose tissue; Brown adipose thermogenesis; Cardiac cachexia; Circadian clock; Fat expenditure; Heart failure; White adipose lipolysis

DOI: https://doi.org/10.1038/s41598-024-58577-8

J Neurosci. 2024 Apr 09. pii: e0813232024. [Epub ahead of print]

Cholinergic neurotransmission controls orexigenic endocannabinoid signaling in the gut in diet-induced obesity.

Courtney P Wood, Camila Alvarez, Nicholas V DiPatrizio.

The brain bidirectionally communicates with the gut to control food intake and energy balance, which becomes dysregulated in obesity. For example, endocannabinoid (eCB) signaling in the small-intestinal epithelium (SI) is upregulated in diet-induced obese mice (DIO) and promotes overeating by a mechanism that includes inhibiting gut-brain satiation signaling. Upstream neural and molecular mechanism(s) involved in overproduction of orexigenic gut eCBs in DIO, however, are unknown. We tested the hypothesis that overactive parasympathetic signaling at muscarinic acetylcholine receptors (mAChRs) in the SI increases biosynthesis of the eCB, 2-arachidonoyl-sn-glycerol (2-AG), which drives hyperphagi-a via local CB1Rs in DIO. Male mice were maintained on a high-fat/high-sucrose western-style diet for 60 days, then administered several mAChR antagonists 30 min prior tissue harvest or a food intake test. Levels of 2-AG and activity of its metabolic enzymes in the SI were quantitated. DIO mice, when compared to those fed a low-fat/no-sucrose diet, displayed increased expression of cFos protein in the dorsal motor nucleus of the vagus, which suggests increased activity of efferent cholinergic neurotransmission. These mice exhibited elevated levels of 2-AG biosynthesis in the SI, which was reduced to control levels by mAChR antagonists. Moreover, the peripherally-restricted mAChR antagonist, methylhomatropine bromide, and the peripherally-restricted CB1R antagonist, AM6545, reduced food intake in DIO mice for up to 24 h but had no effect in mice conditionally deficient in SI CB1Rs. These results suggest that hyperactivity at mAChRs in the periphery increases formation of 2-AG in the SI and activates local CB1Rs, which drives hyperphagia in DIO.Significance Statement Gut-brain signaling controls food intake and energy homeostasis; however, it is poorly understood how gut-brain signaling becomes dysregulated in obesity. In this study, we demonstrated that brain to gut communication is altered in obesity, leading to an increase in endocannabinoid signaling in the GI tract, which drives overeating. Acutely blocking activity at muscarinic acetylcholine receptors in the periphery attenuates intestinal endocannabinoid production and calorie intake in obese animals. This effect was absent in mice conditionally lacking CB1Rs in the intestinal epithelium. These findings expand our understanding of the complex pathophysiology associated with obesity and mechanisms of brain-gut-brain signaling.

DOI: https://doi.org/10.1523/JNEUROSCI.0813-23.2024