Overexpression of Gjb4 impairs cell proliferation and insulin secretion in primary islet cells

Abstract

Objective Altered gene expression contributes to the development of type 2 diabetes (T2D); thus, the analysis of differentially expressed genes between diabetes-susceptible and diabetes-resistant mouse models is an important tool for the determination of candidate genes that participate in the pathology. Based on RNA-seq and array data comparing pancreatic gene expression of diabetes-prone New Zealand Obese (NZO) mice and diabetes-resistant B6.V- ob/ob (B6- ob/ob ) mice, the gap junction protein beta 4 ( Gjb4 ) was identified as a putative novel T2D candidate gene. Methods Gjb4 was overexpressed in primary islet cells derived from C57BL/6 (B6) mice and INS-1 cells via adenoviral-mediated infection. The proliferation rate of cells was assessed by BrdU incorporation, and insulin secretion was measured under low (2.8 mM) and high (20 mM) glucose concentration. INS-1 cell apoptosis rate was determined by Western blotting assessing cleaved caspase 3 levels. Results Overexpression of Gjb4 in primary islet cells significantly inhibited the proliferation by 47%, reduced insulin secretion of primary islets (46%) and INS-1 cells (51%), and enhanced the rate of apoptosis by 63% in INS-1 cells. Moreover, an altered expression of the miR-341-3p contributes to the Gjb4 expression difference between diabetes-prone and diabetes-resistant mice. Conclusions The gap junction protein Gjb4 is highly expressed in islets of diabetes-prone NZO mice and may play a role in the development of T2D by altering islet cell function, inducing apoptosis and inhibiting proliferation.

Characterization of the microRNA Transcriptomes and Proteomics of Cochlea Tissue-derived Small Extracellular Vesicles From Different Age of Mice After Birth

“…The miRNAs miR3074-1-3p, miR3095-3p, miR344b-3p, and miR3057-5p have no reported biological functions. For the decreased miRNAs, miR495-3p [58], miR140-3p [59], miR434-5p [60], miR322-3p [61], miR409-3p [62], miR674-3p [63], miR335-3p [61], miR543-3p [64], miR341-3p [65], miR202-5p [66], miR369-3p [67], miR330-3p [68], miR370-3p [69], miR335-5p [70], miR503-3p [71], and miR503-5p [72] have been reported that related to biological processes, and only miR299a-3p has no reported function. We veri ed the 18 increased miRNAs by qPCR, and Fig.…”

Alternative exon splicing and differential expression in pancreatic islets reveals candidate genes and pathways implicated in early diabetes development

“…Gjb4 , another gene upregulated in NZO, is localized in a T2D QTL on distal chromosome 4 (designated Nbg4d ), associated with improved glycemia in NZO-allele carriers (Schallschmidt et al 2018 ). Recently, overexpression of Gjb4 in primary islets was shown to impair β-cell proliferation and insulin secretion (Gassler et al 2020 ), indicating that Gjb4 functions as a risk gene in T2D development and may thus contribute to the high T2D susceptibility in NZO. However, a causal relationship with our QTL which is associated with a protection from T2D in NZO-allele carriers, is rather unlikely.…”

MiR‐205 is up‐regulated in islets of diabetes‐susceptible mice and targets the diabetes gene Tcf7l2

“…Here, the miR-QTL-Scan, a computational framework, previously described by our group, 7 was adapted to screen for novel miRNAs relevant for islet-cell function. In contrast to previous studies that focused on the most significantly altered miRNAs and/or miRNAs with the highest fold change, 4,30 the current work combined different information layers to select candidates.…”

“…Western blotting was performed as described previously 4,47 using a 12% polyacrylamide gel loaded with 10 μg protein. Primary antibodies against TCF7L2 (1:1000, ab76151, Abcam, Cambridge, UK), PLCB1 (1:1000, ab182359, EPR19085, Abcam) and α-tubulin as loading control (1:1000, Sigma, St. Louis, USA) were used, followed by application of a secondary horseradish 10 peroxidase-conjugated antimouse antibody (1:20 000, Dianova, Hamburg, Germany) and goat peroxidase-conjugated anti-rabbit antibody (1:20 000, Dianova).…”

“…2 In previous studies, we performed a genome-wide linkage analysis with the NZO and C57BL/6 (B6) strains and detected six quantitative trait loci (QTL) 3 that participate in the development of insulin resistance and T2D. The investigation of these QTL allowed not only the identification of genes (Gjb4, Zfp69, Tbc1d1) [4][5][6] involved in this complex trait, but also led to the discovery of small non-coding RNAs, for example, microRNAs (miRNA) that participate in the disease progression. Expression of miR-31, located in the obesity QTL Nob6, was elevated in adipose tissue of obese mice and humans and its manipulation in a human adipocyte cell line further confirmed that miR-31 targets genes involved in adipogenesis and insulin signalling.…”