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Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System
Published on: December 16, 2021
Identification of four trp1 gene variants murine pancreatic beta-cells
1University Laboratory of Physiology, Oxford, UK.
Abstract:
Insulin secretion is stimulated by glucose, hormones and neurotransmitters. Both activation of a non-selective cation current and activation of a Ca2+ current in response to depletion of intracellular Ca2+ stores have been suggested to play a role in this stimulation. The properties of these currents resemble those reported for the Drosophila genes trp and trpl. Using the reverse transcription polymerase chain reaction and Northern blot analysis we found that of the six mammalian trp-related genes (trp1-6), only trp1 was expressed at high levels in the mouse insulinoma cell line MIN6. We cloned the murine homologue of human trp1 from MIN6 cells and identified four variants (alpha, beta, gamma and delta), generated by alternative splicing near the N-terminus of the protein. In vitro translation showed that only the alpha and beta splice variants are efficiently expressed. The beta variant is the dominant form in MIN6 cells (and probably in mouse pancreatic islets), whereas the alpha variant is the major type in the mouse brain. The beta variant showed 99% identity to the human homologue at the amino acid level.
Insights
Researchers identified a specific TRP1 gene variant crucial for insulin secretion. This finding sheds light on the molecular mechanisms regulating glucose-stimulated insulin release in pancreatic cells.
Area of Science:
- Molecular Biology
- Cell Physiology
- Endocrinology
Background:
- Insulin secretion, vital for glucose homeostasis, is modulated by various stimuli.
- Ion channels, including non-selective cation and Ca2+ currents, are implicated in insulin release.
- Mammalian TRP (Transient Receptor Potential) channels share similarities with Drosophila TRP and TRPL proteins.
Purpose of the Study:
- To investigate the role of mammalian TRP genes in insulin secretion.
- To identify specific TRP1 splice variants expressed in pancreatic beta cells.
- To characterize the expression patterns of TRP1 variants in different tissues.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis to detect TRP gene expression.
- Cloning and sequencing of the murine TRP1 gene and its splice variants.
- In vitro translation assays to assess the expression efficiency of different variants.
Main Results:
- Only TRP1 among mammalian TRP genes (TRP1-6) showed high expression in MIN6 insulinoma cells.
- Four TRP1 splice variants (alpha, beta, gamma, delta) were identified in mouse cells.
- TRP1 variants alpha and beta were efficiently expressed in vitro, with beta being dominant in MIN6 cells and pancreatic islets, and alpha in the brain.
- The beta variant exhibited 99% amino acid identity to its human homologue.
Conclusions:
- TRP1 is the predominant mammalian TRP gene expressed in pancreatic beta cells.
- Alternative splicing generates multiple TRP1 variants, with distinct tissue distribution.
- The TRP1 beta variant is a key player in insulin secretion and is highly conserved between mice and humans.
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