Pancreatic a-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human a-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality a-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live a-cells from dissociated human islet cells with 95% purity. The a-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form a-pseudoislets. The a-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key a-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in a-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary a-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.
Publications by Year: 2023
Congenital hyperinsulinism of infancy (CHI) can be caused by deficiency of the ubiquitously expressed enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). To test the hypothesis that SCHAD-CHI arises from a specific defect in pancreatic β-cells, we created genetically engineered β-cell-specific (β-SKO) or hepatocyte-specific (L-SKO) SCHAD knockout mice. Whereas L-SKO mice were normoglycemic, plasma glucose in β-SKO animals was significantly reduced in the random fed state, after overnight fasting, and following refeeding. The hypoglycemic phenotype was exacerbated when the mice were fed a diet enriched in leucine, glutamine and alanine. Intraperitoneal injection of these three amino acids led to rapid elevation in insulin levels in β-SKO mice compared to controls. Consistently, treating isolated β-SKO islets with the amino acid mixture potently enhanced insulin secretion compared to controls in a low-glucose environment. RNA sequencing of β-SKO islets revealed reduced transcription of β-cell identity genes and upregulation of genes involved in oxidative phosphorylation, protein metabolism and Ca2+ handling. The β-SKO mouse offers a useful model to interrogate the intra-islet heterogeneity of amino acid sensing given the very variable expression levels of SCHAD within different hormonal cells, with high levels in β- and δ-cells and virtually absent α-cell expression. We conclude that the lack of SCHAD protein in β-cells results in a hypoglycemic phenotype characterized by increased sensitivity to amino acid-stimulated insulin secretion and loss of β-cell identity.
Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. We recently discovered that neuronal regeneration-related protein (NREP/P311), an epigenetically regulated gene reprogrammed by parental metabolic syndrome, is downregulated in human NAFLD. To investigate the impact of NREP insufficiency, we used RNA-sequencing, lipidomics, and antibody microarrays on primary human hepatocytes. NREP knockdown induced transcriptomic remodeling that overlapped with key pathways impacted in human steatosis and steatohepatitis. Additionally, we observed enrichment of pathways involving phosphatidylinositol signaling and one-carbon metabolism. Lipidomics analyses also revealed an increase in cholesterol esters and triglycerides and decreased phosphatidylcholine levels in NREP-deficient hepatocytes. Signalomics identified calcium signaling as a potential mediator of NREP insufficiency's effects. Our results, together with the encouraging observation that several single nucleotide polymorphisms (SNPs) spanning the NREP locus are associated with metabolic traits, provide a strong rationale for targeting hepatic NREP to improve NAFLD pathophysiology.
Type 1 (T1D) or type 2 diabetes (T2D) are caused by a deficit of functional insulin-producing β cells. Thus, the identification of β cell trophic agents could allow the development of therapeutic strategies to counteract diabetes. The discovery of SerpinB1, an elastase inhibitor that promotes human β cell growth, prompted us to hypothesize that pancreatic elastase (PE) regulates β cell viability. Here, we report that PE is up-regulated in acinar cells and in islets from T2D patients, and negatively impacts β cell viability. Using high-throughput screening assays, we identified telaprevir as a potent PE inhibitor that can increase human and rodent β cell viability in vitro and in vivo and improve glucose tolerance in insulin-resistant mice. Phospho-antibody microarrays and single-cell RNA sequencing analysis identified PAR2 and mechano-signaling pathways as potential mediators of PE. Taken together, our work highlights PE as a potential regulator of acinar-β cell crosstalk that acts to limit β cell viability, leading to T2D.
Monogenic diabetes includes several clinical conditions generally characterized by early-onset diabetes, such as neonatal diabetes, maturity-onset diabetes of the young (MODY) and various diabetes-associated syndromes. However, patients with apparent type 2 diabetes mellitus may actually have monogenic diabetes. Indeed, the same monogenic diabetes gene can contribute to different forms of diabetes with early or late onset, depending on the functional impact of the variant, and the same pathogenic variant can produce variable diabetes phenotypes, even in the same family. Monogenic diabetes is mostly caused by impaired function or development of pancreatic islets, with defective insulin secretion in the absence of obesity. The most prevalent form of monogenic diabetes is MODY, which may account for 0.5-5% of patients diagnosed with non-autoimmune diabetes but is probably underdiagnosed owing to insufficient genetic testing. Most patients with neonatal diabetes or MODY have autosomal dominant diabetes. More than 40 subtypes of monogenic diabetes have been identified to date, the most prevalent being deficiencies of GCK and HNF1A. Precision medicine approaches (including specific treatments for hyperglycaemia, monitoring associated extra-pancreatic phenotypes and/or following up clinical trajectories, especially during pregnancy) are available for some forms of monogenic diabetes (including GCK- and HNF1A-diabetes) and increase patients' quality of life. Next-generation sequencing has made genetic diagnosis affordable, enabling effective genomic medicine in monogenic diabetes.
The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.