Expression of Transient Receptor Potential Channel Genes and Their Isoforms in Alpha-Cells and Beta-Cells of Human Islets of Langerhans

Expression of the transient receptor potential (TRP) channel genes and their isoforms in the alpha-cells and the beta-cells of the human islets of Langerhans has not been studied in detail. In this study, we have analyzed the RNA sequencing data obtained from purified human alpha-cells and beta-cells to identify the genes and their isoforms that are expressed differentially in these two cell types. We found that TRPC1, TRPC4, TRPC7, TRPM3, and TRPML1 were differentially expressed in these two cell types. TRPC1, TRPM3, and TRPML1 were expressed at a higher level in the beta-cells than in the alpha-cells. TRPC4 and TRPC7 were expressed at a higher level in the alpha-cells than in the beta-cells. The TRPC4-206 isoform was expressed at a 45-fold higher level in the alpha-cells compared to the beta-cells. Expression of TRPM3-202 was 200-fold and TRPM3-209 was 25-fold higher in the beta-cells than in the alpha-cells. Our study has demonstrated the relative abundance of expression of the TRP channel genes and their isoforms in the human alpha-cells and the beta-cells.


Introduction
The pancreatic islets of Langerhans contain three major types of cells, the α-, β-, and δ-cells that secrete glucagon, insulin, and somatostatin, respectively [1]. Other minor cell types of islets are the ghrelin-producing ϵ-cells and the pancreatic polypeptide cells [2,3]. The endocrine cells of islets secrete more than just the main peptide hormone products [2,3]. In the human islets, the relative abundance of the α-, β-, and δ-cells is 10-65%, 28-75%, and 1.2-22%, respectively [4]. However, the estimated percentages of different cell types differ considerably between studies [5][6][7]. Impairment in the normal process of secretion of insulin and glucagon from the islet cells is involved in the pathogenesis of diabetes, a major public health problem throughout the world [8]. The molecular mechanisms involved in the secretion of insulin from the β-cells, and glucagon from the αcells include intermediary metabolism of nutrients and par-ticipation of many ion channels [8]. In this respect, the roles of several ion channels of the transient receptor potential (TRP) family have received considerable attention [9][10][11].
We have reported which TRP channel genes are expressed in the human β-cells [12]. In that study, we analyzed RNA-seq (RNA sequencing) data obtained from βcells of only two human donors. The purity of β-cells used in that study was not high. We did not include the TRP channel genes that were expressed at low levels because we used an arbitrary cut-off point as an expression threshold by which we described whether a gene was expressed or not [12]. We examined the expression of the TRP channel genes only in the β-cells, but not in the α-cells.
In this study, we have analyzed the RNA-seq data from highly purified human α-cells and β-cells obtained from seven human donors. The purification of human α-cells and β-cells and the initial RNA-seq analysis were done by Blodgett et al. [13]. We have identified the TRP channel genes, including those that are expressed at low level, in these two main types of cells of human islets of Langerhans. We have also identified the different splice variants of these genes that are expressed in these cells. In addition, we have identified the TRP channel genes and their isoforms that are differentially expressed in these two types of cells.

Materials and Methods
For identifying the TRP channels and their splice variants, we analyzed publicly available RNA-seq data obtained from highly purified human pancreatic α-cells and β-cells. The methods for purification of the human α-cells and βcells and the protocols for RNA-sequencing have been described in detail by Blodgett et al. [13]. In short, islets isolated from adult, healthy, deceased humans were dissociated into single cells, fixed, permeabilized, and stained by anti-insulin, anti-glucagon, and anti-somatostatin antibodies, and highly purified (>97% pure) α-cells and β-cells were sorted by fluorescence activated cell sorter using gating strategies described before [13]. RNA was extracted from 6 α-cell preparations and 7 β-cell preparations from 7 adult donors (5 males, 1 female, and 1 undefined) of variable ages (4-60 years), and body mass index (BMI) (21.5-37 kg/m 2 ). Libraries were constructed by RNA fragmentation, firstand second-strand DNA synthesis, ligation of adaptors, amplification, library validation, and removal of ribosomal RNA. Illumina HiSeq 2000 platform was used for 91 base pair paired-end sequencing and 20 million clean reads per sample were acquired [13].
For analyzing differential expression of the genes and the isoforms, we used EBSeq R-package with a false discovery rate ðFDRÞ < 0:01. Only genes and isoforms with PPDE (posterior probability of differential expression) greater than 0.99 were considered differentially expressed [16].
We used mean TPM cut-off of 0.5 to consider whether a gene is expressed or not in each cell population; mean TPM values < 0:5 were transcriptional noise. By low level expression, we meant TPM values < 3.

Expression of TRP Channel
Genes in the β-Cells. Among the TRPC (transient receptor potential canonical) channel genes, TRPC1 was expressed at high level in all samples of β-cells (Figure 1(a)). TRPC5 was expressed at low levels in 5 out of 7 samples of β-cells. TRPC3 was not expressed in the β-cells. We observed expression of TRPC4 at low level in only 1 sample of β-cells. TRPC6 and TRPC7 were expressed at low levels in only 2 and 1 sample of β-cells, respectively.
Among the TRPM (transient receptor potential melastatin) channel genes the expression of TRPM3, TRPM4, and TRPM7 in the β-cells was high. TRPM6 was expressed at low levels in 5 out of 7 β-cell samples. TRPM5 was expressed at low level in only 2 of 7 β-cell samples. TRPM2 was expressed at low level in all the β-cell samples. TRPM1 and TRPM8 were not expressed in the β-cells.
Among the TRPV (transient receptor potential vanilloid) family, TRPV1, TRPV3, and TRPV6 were expressed in all samples of β-cells. TRPV2 was expressed at low levels in 4 out of 7 β-cell samples. Low-level expression of TRPV4 was observed in 2 out of 7 β-cell samples. TRPV5 was not expressed in the β-cells.

Expression of TRP Channel
Genes in the α-Cells. TRPC1 and TRPC4 were expressed at high levels in all α-cell samples ( Figure 1, top panel). TRPC5 and TRPC7 were expressed at low levels in all α-cells samples. TRPC6 was expressed at low level in 3 of 6 α-cell samples.
TRPM4 and TRPM7 were expressed at high levels in all α-cell samples. TRPM2 and TRPM6 were expressed at low levels in 1-2 α-cell samples. TRPM1, TRPM5, and TRPM8 were not expressed in these cells.
TRPV1, TRPV2, and TRPV3 were expressed in all α-cell samples. TRPV4 was expressed at low level in 3 of 6 α-cell samples. TRPV6 was also expressed at low levels in 4 of 6 α-cell samples. TRPV5 was not expressed in the α-cells.

Discussion
By analyzing RNA-seq data obtained from highly purified αcells and β-cells of human islets of Langerhans, we have identified the TRP channel genes and their isoforms that are differentially expressed in these two cell types. The differentially expressed genes were as follows: TRPC1, TRPC4, TRPC7, TRPM3, and TRPML1. TRPM3, TRPC1, and TRPML1 were expressed at a higher level in the β-cells, whereas TRPC4 and TRPC7 were expressed at a higher level in the α-cells. TRPM3-201, TRPM3-202, TRPM3-205,  TRPM3-207, TRPM3-209, TRPM3-212, and TRPML1-204 isoforms were expressed at a higher level in the β-cells, whereas the TRPC4-206 isoform was expressed at a higher level in the α-cells (Figure 1(b)). Higher level of expression of TRPC1 and TRPM3 in the β-cells is consistent with the roles of these channels in the regulation of insulin secretion [17,18].

TRP Channel Genes in the β-Cells. The β-cells expressed
The expression profile of the TRP channel genes in the β-cells reported in the present study is different from that reported in an earlier report [12]. It was reported that the β-cells do not express TRPM5, TRPM6, TRPV1, TRPV2, TRPV3, TRPV4, and TRPV6 [12]. We found that these genes are expressed, albeit at low level, in at least some β-cell preparations. In the previous study, we analyzed only two β-cell samples; in the present study, we have analyzed seven β-cell samples. In the previous study, we used an arbitrary cutoff value of fragments per kilobase million ðFPKMÞ > 1 as an expression threshold to decide whether a gene is expressed or not. Because of using such a cutoff, genes that were expressed at relatively low levels were stated as "not expressed." In the present study, we have reported the results as transcripts per kilobase Million (TPM) and we did not use any arbitrary cutoff value. Thus, we have identified all the TRP channel genes including the ones that are expressed at low levels in these cells.
TRPM4 and TRPM5 are closely related nonselective cation channels activated by [Ca 2+ ] i . From studies in rodents, it is known that both ion channels are involved in mediating Ca 2+ signaling and insulin secretion [22,[26][27][28]. Consistent with our previous study, we have found that human β-cells express essentially only the TRPM4 suggesting that the TRPM4 channels are more important than the TRPM5 channels in mediating insulin secretion from human β-cells [12]. There are numerous examples of similar differences between the mouse and the human β-cells [11].
Among the thermosensitive TRP channels TRPM3 is most abundant in the β-cells. Expression of TRPM3 in the β-cells was 25-fold higher compared to that in the α-cells. One isoform of TRPM3 (TRPM3-202) was highly expressed in the β-cells (200-fold higher compared to the α-cells). This isoform is highly homologous to the mouse mTRPM3a4 [29]. Another isoform of TRPM3, namely, the TRPM3-209 was expressed at a level 20.8 times higher in the β-cells than in the α-cells. This isoform is homologous to the mouse mTRP3b2 [30]. Previous studies have shown that TRPM3, which is activated by pregnenolone, contribute to insulin secretion from the β-cells by multiple mechanisms [31,32]. The other four thermosensitive TRP channel genes expressed in the β-cells were TRPV1, TRPV3, TRPV2, and TRPM2, but their expression was much lower (<4 TPM) compared to the expression of TRPM3.
Consistent with a previous report, we could not detect expression of TRPA1, TRPV5, TRPM1, and TRPM8 in the human β-cells [12]. It has been reported that TRPA1 channel is involved in insulin secretion from rat and human βcells [33,34], but our results do not support this view since we found that the human β-cells do not express TRPA1. According to one report TRPV5 (also called ECaC1) chan-nel is abundantly expressed in rat β-cells [35], but our results show that TRPV5 is not expressed in human β-cells.

Functional Implications of TRP Channel Gene Expression in the α-Cells.
Only a few studies have investigated the roles of TRP channels in α-cell function. Human α-cells express TRPM4 but not the TRPM5. Activation of TRPM4 channel depolarizes membrane potential, increases cytoplasmic free Ca 2+ concentration and stimulates glucagon secretion [36]. Glucagon secretagogues like arginine vasopressin (AVP) activate a G q -coupled receptor leading to the production of inositol 1,4,5-trisphosphate and release of Ca 2+ from the endoplasmic reticulum (ER). Emptying of the ER triggers Ca 2+ -entry through the store-operated Ca 2+ channels. Ca 2+ activates the TRPM4, and Na + -influx through the TRPM4 channels and depolarizes the membrane potential. This leads to the activation of the voltage gated Ca 2+ channels and further elevation of the cytoplasmic free Ca 2+ concentration, which triggers glucagon release [36]. It should be noted that TRPM4 is expressed in both human α-cells and β-cells and it may be involved in mediating the secretion of both glucagon and insulin [37].
TRPM7 is also expressed at high level in the α-cells. TRPM7 channels are involved in early pancreatic endocrine development as evidenced from the fact that ablation of TRPM7 in mice reduces total α-cell mass [25].

Some General Comments.
This study is based on β-cells and α-cells obtained from nondiabetic donors aged 4-60 years. For understanding the roles of TRP channels in adaptations and dysfunctions of these cells, it will be necessary to obtain such cells from enough donors of different age groups and from donors who have conditions like impaired glucose tolerance or diabetes. Our results show heterogeneity of expression of TRP channel genes, and we speculate that such   Journal of Diabetes Research heterogeneity could be related to the heterogeneity of the endocrine cells of the islets [38]. We have reported the expression of the TRP channel genes only, but it remains unclear whether these genes lead to the expression of proteins and if those proteins form functional ion channels.
More functional studies will be needed to validate the present findings and elaborate the remaining issues further.

Conclusions
We have identified the TRP channel genes that are expressed in the human α-cells and the β-cells. We have found that TRPC1, TRPC4, TRPC7, TRPM3, and TRPML1 are differentially expressed in these two cell types. TRPC4 and TRPC7 are more expressed in the α-cells than in the β-cells, but the functions of these two channels in the α-cells have not been reported. TRPM3-202 was expressed at 200-fold higher level in the β-cells but the regulation of this channel has not been reported. Our findings about the expression patterns of the different TRP channel genes and their isoforms may contribute to the understanding of the physiological roles of these channels in the regulation of hormone secretion from these cells and other functions of these cells.

Data Availability
The study used RNA sequence data published by Blodget et al., and this has been cited as reference [6] in the text.