Type 2 diabetes mellitus is a complex metabolic disease that has reached epidemic proportions in the United States and around the world. This disease is characterized by loss of insulin secretion and eventually destruction of insulin-producing pancreatic beta cells. Controlling type 2 diabetes is often difficult as pharmacological management routinely requires complex therapy with multiple medications, and loses its effectiveness over time. The objective of this study was to explore the effectiveness of a novel, non-pharmacological approach that utilizes the application of ultrasound energy to augment insulin release from rat INS 832/13 beta cells. The cells were exposed to unfocused ultrasound for 5 min at peak intensity of 1 W/cm2 and frequencies of 400 kHz, 600 kHz, 800 kHz and 1 MHz. Insulin release was measured with enzyme-linked immunosorbent assay (ELISA) and cell viability was assessed via trypan blue dye exclusion test. A marked release (approximately 150 ng/106 cells, p < 0.05) of insulin was observed when beta cells were exposed to ultrasound at 400 kHz and 600 kHz as compared to their initial control values, however this release was accompanied with a substantial loss in cell viability. Ultrasound application at frequencies of 800 kHz resulted in 24 ng/106 cells of released insulin (p < 0.05) as compared to its unstimulated base level, while retaining cell viability. Insulin release from beta cells caused by application of 800 kHz ultrasound was comparable to that reported by secretagogue glucose, thus operating within physiological secretory capacity of these cells. Ultrasound has a potential to find an application as a novel and alternative method to current approaches aimed at correcting secretory deficiencies in patients with type 2 diabetes.
BackgroundOur previous studies have indicated that ultrasound can stimulate the release of insulin from pancreatic beta cells, providing a potential novel treatment for type 2 diabetes. The purpose of this study was to explore the temporal dynamics and Ca2+-dependency of ultrasound-stimulated secretory events from dopamine-loaded pancreatic beta cells in an in vitro setup.MethodsCarbon fiber amperometry was used to detect secretion from INS-1832/13 beta cells in real time. The levels of released insulin were also measured in response to ultrasound treatment using insulin-specific ELISA kit. Beta cells were exposed to continuous wave 800 kHz ultrasound at intensities of 0.1 W/cm2, 0.5 W/cm2 and 1 W/cm2 for several seconds. Cell viability tests were done with trypan blue dye exclusion test and MTT analysis.ResultsCarbon fiber amperometry experiments showed that application of 800 kHz ultrasound at intensities of 0.5 and 1 W/cm2 was capable of stimulating secretory events for durations lasting as long as the duration of the stimulus. Furthermore, the amplitude of the detected peaks was reduced by 64% (p < 0.01) when extracellular Ca2+ was chelated with 10 mM EGTA in cells exposed to ultrasound intensity of 0.5 W/cm2. Measurements of released insulin in response to ultrasound stimulation showed complete inhibition of insulin secretion by chelating extracellular Ca2+ with 10 mM EGTA (p < 0.01). Viability studies showed that 800 kHz, 0.5 W/cm2 ultrasound did not cause any significant effects on viability and metabolic activity in cells exposed to ultrasound as compared to sham-treated cells.ConclusionsOur results demonstrated that application of ultrasound was capable of stimulating the release of insulin from pancreatic beta cells in a safe, controlled and Ca2+-dependent manner.
Focused ultrasound are considered to be a promising tool for the treatment of neurological conditions, overcoming the limitations of current neurostimulation techniques in terms of spatial resolution and invasiveness. Much evidence to support the feasibility of ultrasound activation of neurons at the systemic level has already been provided, but to this day, the biophysical mechanisms underlying ultrasound neurostimulation are still widely unknown. In order to be able to establish a clear and robust causality between acoustic parameters of the excitation and neurobiological characteristics of the response, it is necessary to work at the cellular level, or alternatively on very simple animal models. The study reported here responds to three objectives. Firstly, to propose a simple nervous model for the study of the ultrasound neurostimulation phenomenon, associated with a clear and simple experimental protocol. Secondly, to compare the characteristics of this model’s nervous response to ultrasound neurostimulation with its nervous response to mechanical and electrical stimulation. Thirdly, to study the role played by certain acoustic parameters in the success rate of the phenomenon of ultrasound stimulation. The feasibility of generating action potentials (APs) in the giant axons of an earthworm’s ventral nerve cord, using pulsed ultrasound stimuli (f = 1.1 MHz, Ncycles = 175–1150, PRF = 25–125 Hz, Npulses = 20, PA = 2.5–7.3 MPa), was demonstrated. The time of generation (TOG) of APs associated with ultrasound stimulation was found to be significantly shorter and more stable than the TOG associated with mechanical stimulation (p < 0.001). By applying a causal approach to interpret the results of this study, it was concluded that, in this model, the nervous response to focused ultrasound is initiated along the afferent neurons, in between the mechanosensors and the synaptic connections with the giant axons. Additionally, early results are provided, highlighting a trend for the success rate of ultrasound neurostimulation and number of APs triggered per response to increase with increasing pulse repetition frequency (p < 0.05 and p < 0.001, respectively), increasing pulse duration and increasing pulse amplitude.
Ultrasound, along with other types of energy-based methods, has been widely investigated for use in various therapeutic applications because of its ability to stimulate specific biological processes. Many of these processes are mediated by calcium (Ca2+) signaling, thus making modulation of Ca2+ dynamics an evident therapeutic target for energy-based techniques. Various diseases have been associated with abnormal Ca2+ signaling and could therefore benefit from therapeutic approaches trying to regulate the transport of Ca2+ across cell membranes. Here, we review published literature on the use of mechanical, electrical, magnetic and electromagnetic energy in modulating Ca2+ transients with particular emphasis on therapeutic ultrasound. We further provide brief discussions on the role of Ca2+ in living cells and the use of different experimental techniques to determine and measure its contribution to different biological processes. Finally, we explore the benefits, limitations and potential clinical applications of different energy-based modalities that can be utilized in modulation of Ca2+ signaling.
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