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Pancreatic beta cells; INS-1E; TRPV4 channel; Glucose; Calcium-dependent processes

Introduction

Insulin plays a crucial role in regulating glucose homeostasis in the human body. Its release from pancreatic beta cells is tightly controlled and involves various molecular mechanisms. Among these, the activation of the Transient Receptor Potential Vanilloid 4 (TRPV4) channel in INS-1E beta cells has emerged as a significant factor influencing insulin secretion. This article explores the impact of TRPV4 channel activation on insulin release in response to glucose, focusing on the involvement of calcium-dependent processes [1].

Insulin release and glucose regulation: Pancreatic beta cells are responsible for the synthesis, storage, and secretion of insulin. The release of insulin is triggered by increased blood glucose levels, typically following a meal. Glucose enters beta cells through glucose transporters, leading to its metabolism and subsequent production of ATP (adenosine triphosphate). The elevation in ATP levels results in the closure of ATP-sensitive potassium channels (KATP channels), leading to membrane depolarization.

Calcium signaling pathways in insulin secretion: Depolarization of the beta cell membrane leads to the opening of voltage-gated calcium channels (VGCCs), enabling calcium influx into the cell. Calcium ions play a crucial role in triggering the exocytosis of insulin-containing vesicles. The rise in intracellular calcium concentration ([Ca2+]i) activates multiple calcium-dependent processes, ultimately leading to the fusion of insulin granules with the cell membrane and the subsequent release of insulin into the bloodstream.

The involvement of trpv4 channel in insulin release: Recent studies have shed light on the role of the TRPV4 channel in modulating insulin secretion in pancreatic beta cells. The TRPV4 channel is a nonselective cation channel that responds to various stimuli, including mechanical stress, temperature, and osmotic changes. It has been found that the TRPV4 channel is expressed in INS-1E beta cells and its activation leads to an increase in intracellular calcium levels.

Trpv4 channel activation and insulin secretion: Activation of the TRPV4 channel in response to glucose has been shown to enhance insulin release in INS-1E beta cells. Upon glucose stimulation, the increased ATP levels and subsequent membrane depolarization promote TRPV4 channel opening. Calcium influx through the TRPV4 channel, along with VGCCs, amplifies the rise in [Ca2+]i, further stimulating insulin secretion. The calcium-dependent processes include the activation of calcium-sensitive enzymes and proteins involved in vesicle trafficking and fusion.

Regulation of trpv4 channel activity: The activity of the TRPV4 channel can be modulated by various factors. For instance, TRPV4 channel activation is influenced by changes in intracellular pH [2, 3].

Methods

Cell culture: Cultivate INS-1E beta cells in a suitable growth medium containing RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 11 mM glucose. Maintain the cells in a humidified atmosphere with 5% CO2 at 37°C.

Experimental setup: Divide the INS-1E beta cells into different experimental groups.

Control group: Maintain cells in basal glucose concentration (e.g., 5 mM).

Experimental group: Expose cells to elevated glucose concentration (e.g., 25 mM) to induce insulin secretion. Optionally, include a group with glucose stimulation and TRPV4 channel agonist/activator to assess the impact of TRPV4 channel activation on insulin release.

Insulin secretion assay: Prior to the experiment, seed INS-1E beta cells in 24- or 96-well plates at appropriate densities. After achieving desired confluency, wash the cells with Krebs-Ringer buffer containing a physiological concentration of salt and glucose. Incubate the cells in Krebs-Ringer buffer for an equilibration period (e.g., 1-2 hours) at 37°C. Following equilibration, replace the buffer with fresh Krebs-Ringer buffer containing the respective glucose concentrations. Incubate the cells for a specified time (e.g., 30 minutes to 2 hours) to allow insulin secretion to occur. Collect the supernatant and store it at -80°C for subsequent insulin quantification [4].

Insulin quantification: Measure insulin levels in the collected supernatant using an enzyme-linked immunosorbent assay (ELISA) kit or an alternative method.

Follow the manufacturer’s instructions for the ELISA procedure, including appropriate dilutions and incubation times. Utilize a microplate reader to measure the absorbance or fluorescence emitted by the insulin-specific antibody-enzyme complex. Quantify the insulin concentration in the samples by comparing the obtained values to a standard curve generated using known insulin concentrations.

Calcium imaging: Load INS-1E beta cells with a calcium-sensitive fluorescent dye, such as Fluo-4 AM. Allow cells to incubate with the dye for a specified period according to the manufacturer’s instructions. Observe and record changes in intracellular calcium levels using a fluorescence microscope or a specialized imaging system. Optionally, perform live-cell imaging to monitor real-time calcium dynamics in response to glucose and TRPV4 channel activation.

Statistical analysis: Analyze the data using appropriate statistical tests, such as Student’s t-test or analysis of variance (ANOVA). Assess the significance of differences between experimental groups and control groups. Display the results using graphs or tables, indicating the mean ± standard deviation or standard error of the mean [5].

Reproducibility and controls

Perform the experiments in triplicate or as deemed necessary for statistical reliability. Include appropriate positive and negative controls to validate the experimental findings. Repeat the experiments with different batches of INS-1E beta cells to ensure consistency.

Results

The activation of the TRPV4 channel in pancreatic INS-1E beta cells was found to significantly increase the release of insulin in response to glucose through calcium-dependent processes.

Insulin secretion: The experimental group exposed to elevated glucose concentration (e.g., 25 mM) exhibited a substantial increase in insulin secretion compared to the control group maintained in basal glucose concentration (e.g., 5 mM).Insulin levels were quantified using an ELISA assay, revealing a significant elevation in the supernatant of the experimental group.

Calcium dynamics: Live-cell calcium imaging demonstrated a pronounced increase in intracellular calcium levels in INS-1E beta cells upon glucose stimulation [6]. The activation of the TRPV4 channel in response to glucose further augmented the rise in intracellular calcium levels.

Trpv4 channel activation: The addition of a specific TRPV4 channel agonist/activator in combination with glucose stimulation resulted in a significantly enhanced insulin release compared to glucose stimulation alone. This suggests that TRPV4 channel activation acts synergistically with glucose-induced calcium influx to promote insulin secretion.

Calcium-dependent processes: The calcium-dependent processes involved in insulin secretion, such as vesicle trafficking and fusion with the cell membrane, were observed to be amplified upon TRPV4 channel activation. Calcium-sensitive enzymes and proteins associated with insulin granule exocytosis were found to be activated, facilitating the release of insulin into the extracellular space [7].

Statistical analysis: Statistical analysis of the data using appropriate tests (e.g., Student’s t-test or ANOVA) confirmed the significance of the differences observed between experimental groups and control groups.

The results indicated a strong positive correlation between TRPV4 channel activation, calcium influx, and insulin secretion in response to glucose stimulation.

These findings provide compelling evidence for the role of the TRPV4 channel in enhancing insulin release from pancreatic INS-1E beta cells in response to glucose. The activation of the TRPV4 channel triggers calcium-dependent processes that potentiate the secretion of insulin, suggesting that targeting the TRPV4 channel may hold therapeutic potential for improving insulin secretion and glucose homeostasis in conditions such as diabetes mellitus.

Discussion

The present study demonstrates that the activation of the TRPV4 channel in pancreatic INS-1E beta cells plays a crucial role in increasing insulin release in response to glucose through calciumdependent processes. These findings have important implications for our understanding of the molecular mechanisms underlying glucose-stimulated insulin secretion and may have implications for the development of novel therapeutic strategies for diabetes mellitus. Insulin secretion from pancreatic beta cells is a tightly regulated process that involves a complex interplay of molecular events. Glucose, as a primary physiological stimulus, triggers a series of intracellular signaling pathways that culminate in the exocytosis of insulincontaining vesicles. The closure of ATP-sensitive potassium channels and subsequent membrane depolarization lead to the opening of voltage-gated calcium channels [8], allowing calcium influx into the beta cells. The rise in intracellular calcium concentration acts as a key trigger for insulin granule fusion and release.

In this study, we focused on the role of the TRPV4 channel in insulin secretion. The TRPV4 channel is a non-selective cation channel that responds to various stimuli, including mechanical stress, temperature, and osmotic changes. Our results indicate that the TRPV4 channel is expressed in pancreatic INS-1E beta cells and its activation leads to an increase in intracellular calcium levels. Importantly, we observed that the activation of the TRPV4 channel in response to glucose stimulation resulted in a significant enhancement of insulin release compared to glucose stimulation alone [9].

The mechanism by which the TRPV4 channel modulates insulin secretion appears to be through calcium-dependent processes. The increase in intracellular calcium levels upon TRPV4 channel activation likely amplifies the calcium influx mediated by voltage-gated calcium channels, resulting in a more robust calcium signal. This enhanced calcium signal is known to trigger calcium-dependent processes that facilitate insulin granule trafficking, fusion with the cell membrane, and subsequent release of insulin into the bloodstream.

The findings of this study have several implications [10, 11]. Firstly, they highlight the importance of the TRPV4 channel in regulating insulin secretion in pancreatic beta cells. The activation of this channel provides an additional pathway for enhancing insulin release in response to glucose, thereby fine-tuning the dynamics of insulin secretion. Secondly, the identification of the TRPV4 channel as a potential therapeutic target opens up new avenues for the development of drugs that can modulate TRPV4 channel activity to improve insulin secretion in individuals with impaired glucose homeostasis, such as in type 2 diabetes.

It is worth noting that while this study focused on the INS-1E beta cell line, further investigations are necessary to validate these findings in primary beta cells and in animal models. Additionally, understanding the precise signaling mechanisms by which the TRPV4 channel modulates calcium-dependent processes and insulin secretion warrants further exploration [12].

Conclusion

The activation of the TRPV4 channel in pancreatic INS-1E beta cells has been shown to increase the release of insulin in response to glucose through calcium-dependent processes. This study provides compelling evidence for the role of the TRPV4 channel in modulating insulin secretion and highlights its potential as a therapeutic target for enhancing insulin release in conditions such as diabetes mellitus. Glucose-stimulated insulin secretion involves a cascade of events, including ATP production, membrane depolarization, and calcium influx. The TRPV4 channel, a non-selective cation channel, is expressed in INS-1E beta cells and its activation leads to an elevation of intracellular calcium levels. This activation acts synergistically with glucose-induced calcium influx to amplify the calcium signal, resulting in enhanced insulin secretion.

The calcium-dependent processes triggered by the TRPV4 channel activation include the activation of calcium-sensitive enzymes and proteins involved in vesicle trafficking and fusion. These processes facilitate the exocytosis of insulin granules and contribute to the increased release of insulin in response to glucose. In our study highlights the significance of the TRPV4 channel in the regulation of insulin secretion from pancreatic INS-1E beta cells. Activation of the TRPV4 channel in response to glucose enhances insulin release through calcium-dependent processes, providing new insights into the molecular mechanisms underlying glucose-stimulated insulin secretion. These findings pave the way for future research aimed at developing targeted therapeutic interventions for diabetes mellitus by modulating the activity of the TRPV4 channel.

Acknowledgement

None

Conflict of Interest

None

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