Abstract
Type 2 diabetes is a chronic metabolic disease characterized by pancreatic β-cell dysfunction and peripheral insulin resistance. Among individuals with type 2 diabetes, ∼30% exhibit hypomagnesemia. Hypomagnesemia has been linked to insulin resistance through reduced tyrosine kinase activity of the insulin receptor; however, its impact on pancreatic β-cell function is unknown. In this study, through analysis of several single-cell RNA-sequencing data sets in tandem with quantitative PCR validation in both murine and human islets, we identified NIPAL1 (NIPA-like domain containing 1), encoding a magnesium influx transporter, as an islet-enriched gene. A series of immunofluorescence experiments confirmed NIPAL1's magnesium-dependent expression and that it specifically localizes to the Golgi in Min6-K8 cells, a pancreatic β-cell-like cell line (mouse insulinoma 6 clone K8). Under varying magnesium concentrations, NIPAL1 knockdown decreased both basal insulin secretion and total insulin content; in contrast, its overexpression increased total insulin content. Although the expression, distribution, and magnesium responsiveness of NIPAL1 in α-TC6 glucagonoma cells (a pancreatic α-cell line) were similar to the observations in Min6-K8 cells, no effect was observed on glucagon secretion in α-TC6 cells under the conditions studied. Overall, these results suggest that NIPAL1 expression is regulated by extracellular magnesium and that down-regulation of this transporter decreases glucose-stimulated insulin secretion and intracellular insulin content, particularly under conditions of hypomagnesemia.
Highlights
Type 2 diabetes (T2D) is a chronic metabolic disease characterized by pancreatic b-cell dysfunction and peripheral insulin resistance [1]
NIPAL1 was identified as a novel isletenriched protein via a manually curated data mining approach employing several T2D-related scRNA-seq data sets in combination with quantitative real-time PCR (qPCR) validation in both human and murine islets
To understand the functional roles of NIPAL1 in islets, we first examined its expression in a- and b-cell–like cell lines
Summary
A sequential discovery process was designed to identify novel gene transcripts in pancreatic islets (i.e. a- and b-cells) and determine their influence in the secretion of insulin, glucagon, or both (Fig. S1). To characterize the role of NIPAL1 in pancreatic b-cell function, glucose-stimulated insulin secretion (GSIS) assays were conducted in transfected Min6-K8 cells (i.e. NIPAL1 overexpression and knockdown) under both regular and hypo/hypermagnesemic conditions. In GSIS under standard culture conditions (0.8 mM Mg21), neither overexpression nor knockdown of NIPAL1 in Min6-K8 cells showed significant alteration in overall insulin secretion (Fig. 6, A and B). Overexpression of NIPAL1 significantly increased total insulin content under almost every concentration (Fig. 6H) These observations indicate a role for NIPAL1 in insulin secretion and storage in a magnesium-dependent manner. IF experiments of a-TC cells cultured under varying magnesium conditions (Fig. 7E) showed stronger NIPAL1 staining under high Mg21 (5.0 mM), which was confirmed to be significantly increased under mean pixel intensity quantification relative to standard Mg21 (0.8 mM) (Fig. 7F). NIPAL1 may play a role in glycosylation and in the regulation of the Notch signaling pathway (Fig. S3)
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