ND-654

One in ten pregnancies in the US, suffers from Gestational Diabetes Mellitus (GDM). Offspring born to mothers who have GDM have a higher risk of developing Obesity, Type 2 diabetes and Non‐alcoholic fatty liver disease later in life. Although in utero glucose exposure provides evidence for developmental programming leading to metabolic disorders in adults, the molecular mechanisms are still unclear. We aim to understand the mechanisms implicated in developmental programming and their associated adult outcomes in a well‐established zebrafish model of fetal hyperglycemia that mimics the metabolic disorders in human GDM.

We developed a zebrafish model of fetal hyperglycemia by exposing embryos to high glucose (4.5% w/v) during the last day of embryogenesis from 96 to 120 hours post fertilization. Glucose level was detected in embryos and larvae homogenates and in adult serum by glucometer. Body composition was determined in adults by EchoMRI, gene expression of glycolytic, lipogenic (Acetyl‐CoA Carboxylase (acca) and mitochondria biogenesis (Peroxisome proliferator‐activated receptor‐gamma coactivator [ PGC ‐ 1α/β ) markers were quantified by RT‐qPCR and acca by western blot. Lipid accumulation was detected by Oil‐Red‐O staining (ORO) and quantified by spectrophotometry. Triglyceride and cholesterol levels (ELISA), adiposity (Nile red staining) and mitochondrion number (mitotracker staining) were also evaluated. We also assessed if co‐administration of metformin or ND646 (known inhibitors of acca) with glucose can attenuate fetal hyperglycemia‐mediated metabolic outcomes.

Zebrafish embryos exposed to high glucose for 24 hrs are transiently hyperglycemic while normoglycemia is restored after glucose exposure. Larvae and adults with embryonic glucose exposure are normoglycemic. Embryos showed an upregulation of hexokinase and pyruvate Kinase expression when assessed at 120 hpf, but no change in expression was detected in larvae. Embryos exposed to glucose showed increased lipid accumulation by ORO staining (0.22 ±0.01 Vs 0.17±0.01) while larvae showed a 5‐fold increase in adiposity. Adult fish develop obesity characterized by increased BMI (0.83±26.34 Vs. 0.60± 33.89) kg/m 2 and fat mass (12.07± 2.92 Vs 3.4± 0.4) g. Embryos showed increase in both acca mRNA expression (3‐folds) and activation (decreased phosphorylation). Interestingly, acca expression remained increased in larvae while glycemia was normal. Moreover, there was a significant increase in cholesterol (57.92± 0.7 Vs 34.8±1.01 µM) and triglyceride levels in glucose exposed embryos compared to controls (4.1± 0.75 Vs 0.98±0.27 µM). Our data in embryos revealed an increase in mitochondria number validated by an increase in the expression of PGC ‐ 1α/β . Finally, ORO staining showed that embryonic lipid accumulation was decreased to normal levels, by metformin or ND646 exposure when co‐administered with glucose.

Together our results show that embryonic glucose exposure alters developmental programming, including lipid homeostasis dysregulation that is persistent in larvae and adult. However, it can be rescued by pharmacological approaches.

It is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we derive airway organoids from human pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 infection. Using this platform, we perform a high content screen and identify GW6471, which blocks SARS-CoV-2 infection. GW6471 can also block infection of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 infection at least in part by inhibiting hypoxia inducible factor 1 subunit alpha (HIF1α), which is further validated by chemical inhibitor and genetic perturbation targeting HIF1α. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1α-glycolysis axis in mediating SARS-CoV-2 infection of human airway epithelium.