Physical tuning of galectin-3 signaling.

Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3-glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits ("Dimer," "Trimer," "Tetramer," "Pentamer," "Hexamer") demonstrated glycan-binding properties and cell death-inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

A Novel ex vivo Method for Investigating Vascularization of Transplanted Islets.

Revascularization of transplanted pancreatic islets is critical for survival and treatment of type 1 diabetes. Questions concerning how islets influence local microvascular networks and how networks form connections with islets remain understudied and motivate the need for new models that mimic the complexity of real tissue. Recently, our laboratory established the rat mesentery culture model as a tool to investigate cell dynamics involved in microvascular growth. An advantage is the ability to observe blood vessels, lymphatics, and immune cells. The objective of this study was to establish the rat mesentery tissue culture model as a useful tool to investigate islet tissue integration. DiI-labeled islets were seeded onto adult rat mesentery tissues and cultured for up to 3 days. Live lectin labeling enabled time-lapse observation of vessel growth. During culture, DiI-positive islets remained intact. Radial lectin-positive capillary sprouts with DiI labeling were observed to form from islets and connect to host networks. Lectin-positive vessels from host networks were also seen growing toward islets. PECAM and NG2 labeling confirmed that vessels sprouting from islets contained endothelial cells and pericytes. Our results introduce the rat mesentery culture model as a platform for investigating dynamics associated with the initial revascularization of transplanted islets.

Betel quid: New insights into an ancient addiction.

The use of areca nuts (areca) in the form of betel quids constitutes the fourth most common addiction in the world, associated with high risk for oral disease and cancer. Areca is a complex natural product, making it difficult to identify specific components associated with the addictive and carcinogenic properties. It is commonly believed that the muscarinic agonist arecoline is at the core of the addiction. However, muscarinic receptor activation is not generally believed to support drug-taking behaviour. Subjective accounts of areca use include descriptions of both sedative and stimulatory effects, consistent with the presence of multiple psychoactive agents. We have previously reported partial agonism of α4-containing nicotinic acetylcholine receptors by arecoline and subsequent inhibition of those receptors by whole areca broth. In the present study, we report the inhibition of nicotinic acetylcholine receptors and other types of neurotransmitter receptors with compounds of high molecular weight in areca and the ability of low molecular weight areca extract to activate GABA and glutamate receptors. We confirm the presence of a high concentration of GABA and glutamate in areca. Additionally, data also indicate the presence of a dopamine and serotonin transporter blocking activity in areca that could account for the reported stimulant and antidepressant activity. Our data suggest that toxic elements of high molecular weight may contribute to the oral health liability of betel quid use, while two distinct low molecular weight components may provide elements of reward, and the nicotinic activity of arecoline contributes to the physical dependence of addiction.

Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials.

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

Impaired islet function with normal exocrine enzyme secretion is consistent across the head, body, and tail pancreas regions in type 1 diabetes.

Histopathological heterogeneity in human pancreas has been well documented; however, functional evidence at the tissue level is scarce. Herein we investigated in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in no diabetes (ND, n=15), single islet autoantibody-positive (1AAb+, n=7), and type 1 diabetes donors (T1D, <14 months duration, n=5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features were comparable across the regions in ND. In T1D, insulin secretion and beta-cell volume were significantly reduced within all regions, while glucagon and enzymes were unaltered. Beta-cell volume was lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ were consistent across PH, PB and PT. This study supports low inter-regional variation in pancreas slice function and potentially, increased metabolic demand in 1AAb+.

Impaired islet function and normal exocrine enzyme secretion occur with low inter-regional variation in type 1 diabetes.

Histopathological heterogeneity in the human pancreas is well documented; however, functional evidence at the tissue level is scarce. Herein, we investigate in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in donors without diabetes (ND; n = 15), positive for one islet autoantibody (1AAb+; n = 7), and with type 1 diabetes (T1D; <14 months duration, n = 5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features are comparable across regions in ND. In T1D, insulin secretion and beta-cell volume are significantly reduced within all regions, while glucagon and enzymes are unaltered. Beta-cell volume is lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ are consistent across the PH, PB, and PT. This study supports low inter-regional variation in pancreas slice function and, potentially, increased metabolic demand in 1AAb+.

Serum from pregnant donors induces human beta cell proliferation.

Pancreatic beta cells are among the slowest replicating cells in the human body and have not been observed to increase in number except during the fetal and neonatal period, in cases of obesity, during puberty, as well as during pregnancy. Pregnancy is associated with increased beta cell mass to meet heightened insulin demands. This phenomenon raises the intriguing possibility that factors present in the serum of pregnant individuals may stimulate beta cell proliferation and offer insights into expansion of the beta cell mass for treatment and prevention of diabetes. The primary objective of this study was to test the hypothesis that serum from pregnant donors contains bioactive factors capable of inducing human beta cell proliferation. An immortalized human beta cell line with protracted replication (EndoC-ßH1) was cultured in media supplemented with serum from pregnant and non-pregnant female and male donors and assessed for differences in proliferation. This experiment was followed by assessment of proliferation of primary human beta cells. Sera from five out of six pregnant donors induced a significant increase in the proliferation rate of EndoC-ßH1 cells. Pooled serum from the cohort of pregnant donors also increased the rate of proliferation in primary human beta cells. This study demonstrates that serum from pregnant donors stimulates human beta cell proliferation. These findings suggest the existence of pregnancy-associated factors that can offer novel avenues for beta cell regeneration and diabetes prevention strategies. Further research is warranted to elucidate the specific factors responsible for this effect.

Bioartificial matrices for therapeutic vascularization.

Therapeutic vascularization remains a significant challenge in regenerative medicine applications. Whether the goal is to induce vascular growth in ischemic tissue or scale up tissue-engineered constructs, the ability to induce the growth of patent, stable vasculature is a critical obstacle. We engineered polyethylene glycol-based bioartificial hydrogel matrices presenting protease-degradable sites, cell-adhesion motifs, and growth factors to induce the growth of vasculature in vivo. Compared to injection of soluble VEGF, these matrices delivered sustained in vivo levels of VEGF over 2 weeks as the matrix degraded. When implanted subcutaneously in rats, degradable constructs containing VEGF and arginine-glycine-aspartic acid tripeptide induced a significant number of vessels to grow into the implant at 2 weeks with increasing vessel density at 4 weeks. The mechanism of enhanced vascularization is likely cell-demanded release of VEGF, as the hydrogels may degrade substantially within 2 weeks. In a mouse model of hind-limb ischemia, delivery of these matrices resulted in significantly increased rate of reperfusion. These results support the application of engineered bioartificial matrices to promote vascularization for directed regenerative therapies.

Single versus dual microgel species for forming guest-host microporous annealed particle PEG-MAL hydrogel.

Inter-particle secondary crosslinks allow microporous annealed particle (MAP) hydrogels to be formed. Methods to introduce secondary crosslinking networks in MAP hydrogels include particle jamming, annealing with covalent bonds, and reversible noncovalent interactions. Here, we investigate the effect of two different approaches to secondary crosslinking of polyethylene glycol (PEG) microgels via reversible guest-host interactions. We generated a dual-particle MAP-PEG hydrogel using two species of PEG microgels, one functionalized with the guest molecule, adamantane, and the other with the host molecule, ß-cyclodextrin (Inter-MAP-PEG). In a different approach, a mono-particle MAP-PEG hydrogel was generated using one species of microgel functionalized with both guest and host molecules (Intra-MAP-PEG). The Intra-MAP-PEG formed a homogenous distribution due to the single type of microgels used. We then compared the mechanical properties of these two types of MAP-PEG hydrogels and found that Intra-MAP-PEG resulted in significantly softer gels with lower yield stress. We investigated the effect of intra-particle guest-host interactions through titrated weight percentage and the concentration of functional groups added to the hydrogel. We found that there was an ideal concentration of guest-host molecules that enables intra- and inter-particle guest-host interactions with sufficient covalent crosslinking. Based on these studies, Intra-MAP-PEG provides a homogeneous guest-host hydrogel that is shear-thinning with reversible secondary crosslinking.

Granular Hydrogels for Harnessing the Immune Response.

This review aims to understand the current progress in immune-instructive granular hydrogels and identify the key features used as immunomodulatory strategies. Published work is systematically reviewed and relevant information about granular hydrogels used throughout these studies is collected. The base polymer, microgel generation technique, polymer crosslinking chemistry, particle size and shape, annealing strategy, granular hydrogel stiffness, pore size and void space, degradability, biomolecule presentation, and drug release are cataloged for each work. Several granular hydrogel parameters used for immune modulation: porosity, architecture, bioactivity, drug release, cell delivery, and modularity, are identified. The authors found in this review that porosity is the most significant factor influencing the innate immune response to granular hydrogels, while incorporated bioactivity is more significant in influencing adaptive immune responses. Here, the authors' findings and summarized results from each section are presented and suggestions are made for future studies to better understand the benefits of using immune-instructive granular hydrogels.

Immune engineered extracellular vesicles to modulate T cell activation in the context of type 1 diabetes.

Extracellular vesicles (EVs) can affect immune responses through antigen presentation and costimulation or coinhibition. We generated designer EVs to modulate T cells in the context of type 1 diabetes, a T cell-mediated autoimmune disease, by engineering a lymphoblast cell line, K562, to express HLA-A*02 (HLA-A2) alongside costimulatory CD80 and/or coinhibitory programmed death ligand 1 (PD-L1). EVs presenting HLA-A2 and CD80 activated CD8+ T cells in a dose, antigen, and HLA-specific manner. Adding PD-L1 to these EVs produced an immunoregulatory response, reducing CD8+ T cell activation and cytotoxicity in vitro. EVs alone could not stimulate T cells without antigen-presenting cells. EVs lacking CD80 were ineffective at modulating CD8+ T cell activation, suggesting that both peptide-HLA complex and costimulation are required for EV-mediated immune modulation. These results provide mechanistic insight into the rational design of EVs as a cell-free approach to immunotherapy that can be tailored to promote inflammatory or tolerogenic immune responses.

Serum from pregnant donors induces human beta cell proliferation and insulin secretion.

Pancreatic beta cells are among the slowest replicating cells in the human body. Human beta cells usually do not increase in number with exceptions being during the neonatal period, in cases of obesity, and during pregnancy. This project explored maternal serum for stimulatory potential on human beta cell proliferation and insulin output. Gravid, full-term women who were scheduled to undergo cesarean delivery were recruited for this study. A human beta cell line was cultured in media supplemented with serum from pregnant and non-pregnant donors and assessed for differences in proliferation and insulin secretion. A subset of pregnant donor sera induced significant increases in beta cell proliferation and insulin secretion. Pooled serum from pregnant donors also increased proliferation in primary human beta cells but not primary human hepatocytes indicating a cell-type specific effect. This study suggests stimulatory factors in human serum during pregnancy could provide a novel approach for human beta cell expansion.

Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases: Workshop Proceedings.

The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Injectable Microporous Annealed Particle Hydrogel Based on Guest-Host-Interlinked Polyethylene Glycol Maleimide Microgels.

Microporous annealed particle (MAP) hydrogels have emerged as a versatile biomaterial platform for regenerative medicine. MAP hydrogels have been used for the delivery of cells and organoids but often require annealing post injection by an external source. We engineered an injectable, self-annealing MAP hydrogel with reversible interparticle linkages based on guest-host functionalized polyethylene glycol maleimide (PEG-MAL) microgels. We evaluated the effect of guest-host linkages on different types of microgels fabricated by either batch emulsion or mechanical fragmentation methods. Batch emulsion generated small spherical microgels with controllable 10-100 µm diameters and mechanical fragmentation generated irregular microgels with larger diameters (100-200 µm). Spherical microgels (15 µm) showed self-healing behavior and completely recovered from high strain while fragmented microgels (133 µm) did not recover. Guest-host interactions significantly contributed to the mechanical properties of spherical microgels but had no effect on fragmented microgels. Spherical microgels were superior to the fragmented microgels for co-injection of immune cells and pancreatic islets due to their lower force of injection, demonstrating more homogeneously distributed cells and greater cell viability after injection. Based on these studies, the spherical guest-host MAP hydrogels provide a controllable, injectable scaffold for engineered microenvironments and cell delivery applications.

The role of GABA in islet function.

Gamma aminobutyric acid (GABA) is a non-proteinogenic amino acid and neurotransmitter that is produced in the islet at levels as high as in the brain. GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD), of which the 65 kDa isoform (GAD65) is a major autoantigen in type 1 diabetes. Originally described to be released via synaptic-like microvesicles or from insulin secretory vesicles, beta cells are now understood to release substantial quantities of GABA directly from the cytosol via volume-regulated anion channels (VRAC). Once released, GABA influences the activity of multiple islet cell types through ionotropic GABAA receptors and metabotropic GABAB receptors. GABA also interfaces with cellular metabolism and ATP production via the GABA shunt pathway. Beta cells become depleted of GABA in type 1 diabetes (in remaining beta cells) and type 2 diabetes, suggesting that loss or reduction of islet GABA correlates with diabetes pathogenesis and may contribute to dysfunction of alpha, beta, and delta cells in diabetic individuals. While the function of GABA in the nervous system is well-understood, the description of the islet GABA system is clouded by differing reports describing multiple secretion pathways and effector functions. This review will discuss and attempt to unify the major experimental results from over 40 years of literature characterizing the role of GABA in the islet.

Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I.

There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.

Extracellular vesicles in ß cell biology: Role of lipids in vesicle biogenesis, cargo, and intercellular signaling.

BACKGROUND: Type 1 diabetes (T1D) is a complex autoimmune disorder whose pathogenesis involves an intricate interplay between ß cells of the pancreatic islet, other islet cells, and cells of the immune system. Direct intercellular communication within the islet occurs via cell surface proteins and indirect intercellular communication has traditionally been seen as occurring via secreted proteins (e.g., endocrine hormones and cytokines). However, recent literature suggests that extracellular vesicles (EVs) secreted by ß cells constitute an additional and biologically important mechanism for transmitting signals to within the islet. SCOPE OF REVIEW: This review summarizes the general mechanisms of EV formation, with a particular focus on how lipids and lipid signaling pathways influence their formation and cargo. We review the implications of EV release from ß cells for T1D pathogenesis, how EVs and their cargo might be leveraged as biomarkers of this process, and how EVs might be engineered as a therapeutic candidate to counter T1D outcomes. MAJOR CONCLUSIONS: Islet ß cells have been viewed as initiators and propagators of the cellular circuit giving rise to autoimmunity in T1D. In this context, emerging literature suggests that EVs may represent a conduit for communication that holds more comprehensive messaging about the ß cells from which they arise. As the field of EV biology advances, it opens the possibility that intervening with EV formation and cargo loading could be a novel disease-modifying approach in T1D.

Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases: Workshop Proceedings.

ABSTRACT: The "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Guest-host interlinked PEG-MAL granular hydrogels as an engineered cellular microenvironment.

We report the development of a polyethylene glycol (PEG) hydrogel scaffold that provides the advantages of conventional bulk PEG hydrogels for engineering cellular microenvironments and allows for rapid cell migration. PEG microgels were used to assemble a densely packed granular system with an intrinsic interstitium-like negative space. In this material, guest-host molecular interactions provide reversible non-covalent linkages between discrete PEG microgel particles to form a cohesive bulk material. In guest-host chemistry, different guest molecules reversibly and non-covalently interact with their cyclic host molecules. Two species of PEG microgels were made, each with one functional group at the end of the four arm PEG-MAL functionalized using thiol click chemistry. The first was functionalized with the host molecule ß-cyclodextrin, a cyclic oligosaccharide of repeating d-glucose units, and the other functionalized with the guest molecule adamantane. These two species provide a reversible guest-host interaction between microgel particles when mixed, generating an interlinked network with a percolated interstitium. We showed that this granular configuration, unlike conventional bulk PEG hydrogels, enabled the rapid migration of THP-1 monocyte cells. The guest-host microgels also exhibited shear-thinning behavior, providing a unique advantage over current bulk PEG hydrogels.

Effects of sustained GABA releasing implants on pancreatic islets in mice.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that is strongly and selectively synthesized in and secreted from pancreatic beta cells. Exogenously delivered GABA has been proposed to induce beta cell regeneration in type 1 diabetes, but these results have been difficult to replicate and may depend on the specifics of the animal model and drug delivery method used. Here, we developed a GABA-releasing ethylene-vinyl acetate polymer implant for sustained GABA delivery to the intraperitoneal space as an alternative to injected or oral GABA. We explored the effect of the GABA-releasing polymer implants compared to implanted osmotic pumps loaded with GABA on islet size in non-diabetic, outbred mice. We also attempted to monitor in vivo GABA release using HPLC on blood samples, but these measurements were confounded by high variability within treatment groups and unexpectedly high serum GABA levels in mice receiving GABA-negative implants. The ethylene-vinyl acetate polymer implants became heavily fibrosed with abdominal adhesion tissue, while the osmotic pumps had no macroscopic fibrosis. Histological analysis showed no significant effect of the sustained GABA delivery polymer or osmotic pumps on islet size, alpha cell to beta cell ratio, or the number of Ki67-positive islet cells. The GABA treatment time course was limited to two weeks due to the drug-release window of the polymer, while others reported islet-trophic effects of GABA after 10 to 12 weeks of treatment. In summary, our study is consistent with the concept that exogenous GABA administration does not significantly alter islet cell mass in non-diabetic CD-1 mice in the short-term. However, more data are needed including higher GABA doses and more prolonged treatment regimens for a better comparison with contrasting reports.