Biology subjects

Benninger, R. K.

Publications and source records attributed to Benninger, R. K..

5 recordsLinked to original sources

bioRxiv2020

Contrast-enhanced ultrasound measurement of pancreatic blood flow dynamics predicts type1 diabetes therapeutic reversal in preclinical models

In type 1 diabetes (T1D) immune-cell infiltration into the islets of Langerhans (insulitis) and {beta}-cell decline occurs many years before diabetes presents. Non-invasively detecting insulitis and {beta}-cell decline would allow diagnosis of eventual diabetes and provide a means to monitor the efficacy of therapeutic intervention. However, there is a lack of validated clinical approaches for non-invasively imaging disease progression leading to T1D. Islets have a dense microvasculature that reorganizes during diabetes. We previously demonstrated contrast-enhanced ultrasound measurements of pancreatic blood-flow dynamics could predict disease progression in T1D pre-clinical models. Here we test whether these measurements can predict successful therapeutic prevention of T1D. We performed destruction-reperfusion measurements using a small-animal ultrasound machine and size-isolated microbubbles, in NOD-scid mice receiving an adoptive transfer of diabetogenic splenocytes (AT mice). Mice received vehicle control or either of the following treatments: 1) antiCD4 to deplete CD4+ T cells; 2) antiCD3 to block T cell activation, 3) Verapamil to reduce {beta}-cell apoptosis and 4) TUDCA to reduce ER stress. We compared measurements of pancreas blood-flow dynamics with subsequent progression to diabetes. In AT mice blood-flow dynamics were altered >2 weeks after splenocyte transfer. AntiCD4, antiCD3 and verapamil provided a significant delay in diabetes development. Treated AT mice with delayed or absent diabetes development showed significantly altered blood flow dynamics compared to untreated AT mice. Conversely, treated AT mice that developed diabetes, despite therapy, showed similar blood-flow dynamics to untreated AT mice. Thus, contrast-enhanced ultrasound measurement of pancreas blood-flow dynamics can predict the successful or unsuccessful delay or prevention of diabetes upon therapeutic treatments that target both immune activity or {beta}-cell protection. This strategy may provide a clinically deployable predictive marker for disease progression and therapeutic reversal in asymptomatic T1D.

bioengineering↗

bioRxiv2020

Caloric Restriction recovers impaired β-cell-β-cell coupling, calcium oscillation coordination and insulin secretion in prediabetic mice

Caloric restriction has been shown to decrease the incidence of metabolic diseases such as obesity and type 2 diabetes mellitus (T2DM). The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis and are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine if caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high fat diet (HFD), showing indications of prediabetes after 2 months, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to one month of 40% caloric restriction (2g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+ oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+ dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

physiology↗

bioRxiv2020

Engineering Functional Pseudo-Islets of Defined Sizes from Primary Murine Cells Using PEG Microwell Devices

A major limitation of islet transplantation as a therapy for treating Type 1 Diabetes is eventual graft failure, which can be partially attributed to islet cell death. When cultured in vitro, cells in the center of large islets show increased necrosis and exhibit decreased viability and insulin secretion compared to smaller islets. Given the necessity of {beta}-cell-to-{beta}-cell coupling for the physiological response to glucose, a technique to re-aggregate primary islet cells or cells derived from progenitor cells into small clusters of defined sizes may prove advantageous for promoting function upon transplantation. Here, hydrogel microwell arrays were utilized to generate 3-dimensional pseudo-islets from primary murine islets. Pseudo-islets ranged from 50 to 100 m in diameter as controlled through the microwell dimensions, and contained {beta}-, -, and {delta}-cells with ratios similar to those in whole murine islets. Over two weeks in culture, pseudo-islets remained highly viable and responsive to glucose. Intracellular calcium flux showed more robust and coordinated dynamics at high glucose and decreased activity at low glucose compared to age-matched wild-type islets. Therefore, microwell devices can control the aggregation of cells isolated from primary islets to produce islet-like clusters that are functionally similar to freshly isolated islets, and may provide a technique to create improved cellular therapies for Type 1 Diabetes.

bioengineering↗

bioRxiv2019

Optogenetic Stimulation of Pancreatic Function via Vagal Cholinergic Axons

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion; concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools enable high specificity in neural stimulation using cell-type specific targeting of opsins and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting vagal parasympathetic axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to both ultrasound image-guided optical stimulation of axon terminals in the pancreas and optical stimulation of axons of the cervical vagus nerve, together with ultrasound-based measures of pancreas blood flow. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and vagal stimulation, accompanying rapid reductions in glycemic levels. Additionally, a significant increase in pancreatic blood flow was measured following optical stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest therapeutic potential for the control of insulin secretion and glucose homeostasis.

bioengineering↗

bioRxiv2019

How heterogeneity in glucokinase and gap junction coupling determines the islet electrical response

Understanding how cell sub-populations in a tissue impact the function of the overall system is often challenging. There is extensive heterogeneity among insulin-secreting {beta}-cells within islets of Langerhans, including their insulin secretory response and gene expression profile; and this heterogeneity can be altered in diabetes. Several studies have identified variations in nutrient sensing between {beta}-cells, including glucokinase (GK) levels, mitochondrial function or expression of genes important for glucose metabolism. Sub-populations of {beta}-cells with defined electrical properties can disproportionately influence islet-wide free-calcium activity ([Ca2+]) and insulin secretion, via gap junction electrical coupling. However, it is poorly understood how sub-populations of {beta}-cells with altered glucose metabolism may impact islet function. To address this, we utilized a multicellular computational model of the islet in which a population of cells deficient in GK activity and glucose metabolism was imposed on the islet, or where {beta}-cells were heterogeneous in glucose metabolism and GK kinetics were altered. This included simulating Glucokinase gene (GCK) mutations that cause monogenic diabetes. We combined these approaches with experimental models in which gck was genetically deleted in a population of cells or GK was pharmacologically inhibited. In each case we modulated gap junction electrical coupling. Both the simulated islet and the experimental system required 30-50% of the cells to have near-normal glucose metabolism. Below this number, the islet lacked any glucose-stimulated [Ca2+] elevations. In the absence of electrical coupling the change in [Ca2+] was more gradual. As such, given heterogeneity in glucose metabolism, electrical coupling allows a large minority of cells with normal glucose metabolism to promote glucose-stimulated [Ca2+]. If insufficient numbers of cells are present, which we predict can be caused by a subset of GCK mutations that cause monogenic diabetes, electrical coupling exacerbates [Ca2+] suppression. This demonstrates precisely how heterogeneous {beta}-cell populations interact to impact islet function.\n\nSIGNIFICANCEBiological tissues contain heterogeneous populations of cells. Insulin-secreting {beta}-cells within the islets of Langerhans are critical for regulating blood glucose homeostasis. {beta}-cells are heterogeneous but it is unclear how the islet response is impacted by different cell populations and their interactions. We use a multicellular computational model and experimental systems to predict and quantify how cellular populations defined by varied glucose metabolism interact via electrical communication to impact islet function. When glucose metabolism is heterogeneous, electrical coupling is critical to promote electrical activity. However, when cells deficient in glucose metabolism are in the majority, electrical activity is completely suppressed. Thus modulating electrical communication can promotes islet electrical activity, following dysfunction caused by gene mutations that impact glucose metabolism.

biophysics↗