Biology subjects

Shun-Shion, A. S.

Publications and source records attributed to Shun-Shion, A. S..

3 recordsLinked to original sources

bioRxiv2022

Oxygen is a critical regulator of cellular metabolism and function in cell culture

Cell culture is generally considered to be hyperoxic. However, the importance of cellular oxygen consumption is often underappreciated, with rates of oxygen consumption often sufficient to cause hypoxia at cell monolayers. We initially focused on cultured adipocytes as a terminally differentiated cell-type with substantial oxygen consumption rates to support diverse cellular functions. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increasing oxygen diverted glucose flux toward mitochondria and resulted in thousands of gene expression changes that pointed toward alleviated physiological transcriptional responses to hypoxia. Phenotypically, providing more oxygen increased adipokine secretion and rendered adipocytes more sensitive to insulin and lipolytic stimuli. The functional benefits of increasing pericellular oxygen were transferable to other cellular systems including hPSC-derived hepatocytes and cardiac organoids. Our findings suggest that oxygen is limiting in many terminally-differentiated cell culture systems, and that controlling oxygen availability can improve the quality and translatability of cell models.

cell biology↗

bioRxiv2022

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance

The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employed phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults triggering insulin resistance, we observed marked rewiring of the insulin signaling network. This included both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying signaling changes common to multiple insults revealed subnetworks likely containing causal drivers of insulin resistance. Focusing on defective GSK3 signaling initially observed in a relatively small subset of well-characterized substrates, we employed a pipeline for identifying context-specific kinase substrates. This facilitated robust identification of widespread dysregulated GSK3 signaling. Pharmacological inhibition of GSK3 partially reversed insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that encompasses dysregulated GSK3 activity.

molecular biology↗

bioRxiv2022

The exocyst complex is an essential component of the mammalian constitutive secretory pathway

Secreted proteins fulfil a vast array of different functions, including adaptive immunity, cell signalling and extracellular matrix remodelling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane to deliver their contents to the extracellular space. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR knock-outs to study the biosynthetic sorting route from the Golgi apparatus to the plasma membrane. Depletion of core-exocyst subunits reduces carrier fusion and causes cargo accumulation in the post-Golgi carriers. Exocyst subunits co-localise with carriers fusing at the plasma membrane and we show that the exocyst complex is recruited directly to these carriers. Abrogation of exocyst followed by kinetic trafficking assays with multiple different soluble cargoes results in cargo accumulation of all tested cargoes. Unbiased secretomics reveals a drastic reduction in the secretion of soluble proteins to the extracellular milieu after knock-out of exocyst subunits. Importantly, the knock-out of exocyst subunits in specialised secretory cell types prevents the constitutive secretion of antibodies in lymphocytes and of the hormones leptin and adiponectin in adipocytes. Together these data identify the exocyst complex as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.

cell biology↗