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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Nanyang Technological University

Singapore General Hospital

Insulin resistance and β-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and β-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus. 

Apolipoprotein CIII links islet insulin resistance to β-cell failure in diabetes.

The human genome project and its search for factors underlying human diseases has fostered a major human research effort. Therefore, unsurprisingly, in recent years we have observed an increasing number of studies on human islet cells, including disease approaches focusing on type 1 and type 2 diabetes. Yet, the field of islet and diabetes research relies on the legacy of rodent-based investigations, which have proven difficult to translate to humans, particularly in type 1 diabetes. Whole islet physiology and pathology may differ between rodents and humans, and thus a comprehensive cross-species as well as species-specific view on islet research is much needed. In this review we summarise the current knowledge of interspecies islet cytoarchitecture, and discuss its potential impact on islet function and future perspectives in islet pathophysiology research. 

New insights into the architecture of the islet of Langerhans: a focused cross-species assessment.

In vivo imaging of the developing pancreas is challenging due to the inaccessibility of the tissue. To circumvent this, on embryonic day 10.5 (E10.5) we transplanted a mouse developing pancreatic bud into the anterior chamber of the eye (ACE) to determine whether the eye is a useful transplant site to support pancreas development.

The anterior chamber of the eye is a transplantation site that supports and enables visualisation of beta cell development in mice.

Single-cell RNA-seq (scRNA-seq) of pancreatic islets have reported on α- and β-cell gene expression in mice and subjects of predominantly European ancestry. We aimed to assess these findings in East-Asian islet-cells. 448 islet-cells were captured from three East-Asian non-diabetic subjects for scRNA-seq. Hierarchical clustering using pancreatic cell lineage genes was used to assign cells into cell-types. Differentially expressed transcripts between α- and β-cells were detected using ANOVA and in silico replications of mouse and human islet cell genes were performed. We identified 118 α, 105 β, 6 δ endocrine cells and 47 exocrine cells. Besides INS and GCG, 26 genes showed differential expression between α- and β-cells. 10 genes showed concordant expression as reported in rodents, while FAM46A was significantly discordant. Comparing our East-Asian data with data from primarily European subjects, we replicated several genes implicated in nuclear receptor activations, acute phase response pathway, glutaryl-CoA/tryptophan degradations and EIF2/AMPK/mTOR signaling. Additionally, we identified protein ubiquitination to be associated among East-Asian β-cells. We report on East-Asian α- and β-cell gene signatures and substantiate several genes/pathways. We identify expression signatures in East-Asian β-cells that perhaps reflects increased susceptibility to cell-death and warrants future validations to fully appreciate their role in East-Asian diabetes pathogenesis.

Single-cell transcriptomics of East-Asian pancreatic islets cells.

Blood flow regulation in pancreatic islets is critical for function but poorly understood. Here, we establish an in vivo imaging platform in a non-human primate where islets transplanted autologously into the anterior chamber of the eye are monitored non-invasively and longitudinally at single-cell resolution. Engrafted islets were vascularized and innervated and maintained the cytoarchitecture of in situ islets in the pancreas. Blood flow velocity in the engrafted islets was not affected by increasing blood glucose levels and/or the GLP-1R agonist liraglutide. However, islet blood flow was dynamic in nature and fluctuated in various capillaries. This was associated with vasoconstriction events resembling a sphincter-like action, most likely regulated by adrenergic signaling. These observations suggest a mechanism in primate islets that diverts blood flow to cell regions with higher metabolic demand. The described imaging technology applied in non-human primate islets may contribute to a better understanding of human islet pathophysiology.

Article	Year
Apolipoprotein CIII links islet insulin resistance to β-cell failure in diabetes. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25941406	2015
New insights into the architecture of the islet of Langerhans: a focused cross-species assessment. Diabetologia\| PubMed ID: 26215305	2015
The anterior chamber of the eye is a transplantation site that supports and enables visualisation of beta cell development in mice. Diabetologia\| PubMed ID: 26847769	2016
Single-cell transcriptomics of East-Asian pancreatic islets cells. Scientific reports\| PubMed ID: 28694456	2017
Pancreatic Islet Blood Flow Dynamics in Primates. Cell reports\| PubMed ID: 28793270	2017

Yusuf Ali

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