Results: Body weight and triglycerides profiles in blood and liver were comparable between vehicle-and Abn-CBD-treated DIO mice. However, treatment with Abn-CBD reduced hyperinsulinemia and markers of systemic low-grade inflammation in plasma and fat, also promoting white adipose tissue browning. Pancreatic islets from Abn-CBD-treated mice showed lower apoptosis, inflammation and oxidative stress than vehicle-treated DIO mice, and beta cell proliferation was induced. Furthermore, Abn-CBD lowered hepatic fibrosis, inflammation and macrophage infiltration in the liver when compared to vehicle-treated DIO mice. Importantly, the balance between hepatocyte proliferation and apoptosis was improved in Abn-CBD-treated compared to vehicletreated DIO mice. Romero-Zerbo et al. Abn-CBD in Prediabetes and NAFLD Conclusions: These results suggest that Abn-CBD exerts beneficial immunomodulatory actions in the liver, pancreas and adipose tissue of DIO prediabetic mice with NAFLD, thus protecting tissues. Therefore, Abn-CBD and related compounds could represent novel pharmacological strategies for managing obesity-related metabolic disorders.

Hepatocytes were the first cell-type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a non-invasive manner due to the optical inaccessibility of the mammalian liver. Here we take advantage of the transparency of the zebrafish larvae to develop a setup that allows in vivo imaging of calcium flux in zebrafish hepatocytes at cellular resolution. Using this, we provide quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo, which are lost upon starvation. Feeding recommences calcium waves in the liver, but in a spatially restricted manner. Further, ethanol treatment as well as cell ablation induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, while the latter leads to a single calcium spike. Overall, we demonstrate the presence of oscillations, waves and spikes in vivo. Thus, our study introduces a platform for observing diverse calcium dynamics while maintaining the native environment of the liver, which will help investigations into the dissection of molecular mechanisms supporting the intra-and intercellular calcium signaling in the liver.

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover heterogeneity in the thyrocyte population and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line that monitors pax2a expression in the thyrocytes. A population of pax2a-low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a-high and pax2a-low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population.