Enhancing Therapeutic Insulin Transport from Macroencapsulated Islets Using Sub-Minute Pressure at Physiological Levels.

Cadaveric islet and stem cell-derived transplantations hold promise as treatments for type 1 diabetes. To tackle the issue of immunocompatibility, numerous cellular macroencapsulation techniques have been developed that utilize diffusion to transport insulin across an immunoisolating barrier. However, despite several devices progressing to human clinical trials, none have successfully managed to attain physiologic glucose control or insulin independence. Based on empirical evidence, macroencapsulation methods with multilayered, high islet surface density are incompatible with homeostatic, on-demand insulin delivery and physiologic glucose regulation, when reliant solely on diffusion. An additional driving force is essential to overcome the distance limit of diffusion. In this study, we present both theoretical proof and experimental validation that applying pressure at levels comparable to physiological diastolic blood pressure significantly enhances insulin flux across immunoisolation membranes-increasing it by nearly three orders of magnitude. This significant enhancement in transport rate allows for precise, sub-minute regulation of both bolus and basal insulin delivery. By incorporating this technique with a pump-based extravascular system, we demonstrate the ability to rapidly reduce glucose levels in diabetic rodent models, effectively replicating the timescale and therapeutic effect of subcutaneous insulin injection or infusion. This advance provides a potential path towards achieving insulin independence with islet macroencapsulation. One Sentence Summary: Towards improved glucose control, applying sub-minute pressure at physiological levels enhances therapeutic insulin transport from macroencapsulated islets.

Caution on the Use of 68Ga-DOTATATE for the Diagnosis of Pheochromocytoma: A Report of 2 Cases.

Pheochromocytomas are intra-adrenal sympathetic neuroendocrine tumors that arise from chromaffin cells. Paragangliomas similarly arise from chromaffin cells, although at extra-adrenal sites such as sympathetic paraganglia in the abdomen/thorax, or parasympathetic paraganglia in the head/neck. Collectively, pheochromocytomas and paragangliomas are important to diagnose and resect because they may secrete harmful levels of catecholamines, have mass effects, hemorrhage, and/or metastasize. Anatomic imaging of pheochromocytomas is usually completed with computed tomography or magnetic resonance imaging; however, functional imaging may be used to provide additional localization, staging, and/or biologic information. Accordingly, selection of the proper functional imaging modality can be critical to developing the optimal therapeutic strategy. 68Gallium- and 64Copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-octreotate positron emission tomography computed tomography (68Ga- and 64Cu-DOTATATE) are widely used in evaluating pheochromocytomas and paragangliomas, although data regarding the sensitivity for diagnosing pheochromocytoma are limited. We report 2 cases of pheochromocytoma that showed nondiagnostic 68Ga-DOTATATE uptake but were subsequently visualized using alternative functional imaging modalities. Additionally, we provide a review of the literature to highlight the underappreciated limitations of functional adrenal imaging with somatostatin-based compounds.

On-demand electrochemically controlled compound release from an ultrasonically powered implant.

On-demand drug delivery systems are promising for a wide range of therapeutic applications. When combined with wireless implants for controlled drug delivery, they can reduce overall dosage and side effects. Here, we demonstrate release of fluorescein from a novel on-demand release system for negatively charged compounds. The release system is based on a modified electroresponsive polypyrrole nanoparticulate film designed to minimize ion exchange with the stored compound - a major passive leakage mechanism. We further designed an ultrasonically powered mm-sized implant to electronically control the on-demand drug delivery system in vivo. Release kinetics are characterized both in vitro and in vivo in mice using fluorescein as a model drug, demonstrating the feasibility of wireless, controllable drug release using an ultrasonically powered implant.

ß-Cell Succinate Dehydrogenase Deficiency Triggers Metabolic Dysfunction and Insulinopenic Diabetes.

Mitochondrial dysfunction plays a central role in type 2 diabetes (T2D); however, the pathogenic mechanisms in pancreatic ß-cells are incompletely elucidated. Succinate dehydrogenase (SDH) is a key mitochondrial enzyme with dual functions in the tricarboxylic acid cycle and electron transport chain. Using samples from human with diabetes and a mouse model of ß-cell-specific SDH ablation (SDHBßKO), we define SDH deficiency as a driver of mitochondrial dysfunction in ß-cell failure and insulinopenic diabetes. ß-Cell SDH deficiency impairs glucose-induced respiratory oxidative phosphorylation and mitochondrial membrane potential collapse, thereby compromising glucose-stimulated ATP production, insulin secretion, and ß-cell growth. Mechanistically, metabolomic and transcriptomic studies reveal that the loss of SDH causes excess succinate accumulation, which inappropriately activates mammalian target of rapamycin (mTOR) complex 1-regulated metabolic anabolism, including increased SREBP-regulated lipid synthesis. These alterations, which mirror diabetes-associated human ß-cell dysfunction, are partially reversed by acute mTOR inhibition with rapamycin. We propose SDH deficiency as a contributing mechanism to the progressive ß-cell failure of diabetes and identify mTOR complex 1 inhibition as a potential mitigation strategy.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

Inherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

Novel Pathogenic De Novo INS p.T97P Variant Presenting With Severe Neonatal DKA.

Pathogenic INS gene mutations are causative for mutant INS-gene-induced diabetes of youth (MIDY). We characterize a novel de novo heterozygous INS gene mutation (c.289A>C, p.T97P) that presented in an autoantibody-negative 5-month-old male infant with severe diabetic ketoacidosis. In silico pathogenicity prediction tools provided contradictory interpretations, while structural modeling indicated a deleterious effect on proinsulin folding. Transfection of wildtype and INS p.T97P expression and luciferase reporter constructs demonstrated elevated intracellular mutant proinsulin levels and dramatically impaired proinsulin/insulin and luciferase secretion. Notably, proteasome inhibition partially and selectively rescued INS p.T97P-derived luciferase secretion. Additionally, expression of INS p.T97P caused increased intracellular proinsulin aggregate formation and XBP-1s protein levels, consistent with induction of endoplasmic reticulum stress. We conclude that INS p.T97P is a newly identified pathogenic A-chain variant that is causative for MIDY via disruption of proinsulin folding and processing with induction of the endoplasmic reticulum stress response.

Protocol for determining zinc-dependent ß cell-selective small-molecule delivery in mouse pancreas.

Targeted drug delivery to pancreatic islet ß cells is an unmet clinical need. ß cells possess a uniquely high Zn2+ concentration, and integrating Zn2+-binding activity into a small molecule can bias drug accumulation and activity toward ß cells. This protocol can be used to evaluate a molecule's capacity to chelate islet Zn2+, accumulate in islets, and stimulate ß cell-selective replication in mouse pancreas. One obstacle is establishing an LC-MS/MS-based method for compound measurement. Limitations include target compound ionizability and the time-sensitive nature of some experimental assay steps. For complete details on the use and execution of this protocol, please refer to Horton et al. (2019).

Intracardiac paragangliomas: surgical approach and perioperative management.

Intracardiac paragangliomas most commonly arise from the left atrium and are often infiltrative and densely adherent to surrounding structures. Given their rarity, only scattered reports exist in the literature and standardized perioperative and surgical management is not well established. We describe a case of a 60-year-old woman with a mildly functioning intracardiac paraganglioma in which division of the superior vena cava improved exposure and enabled a complex limited resection. Further, we provide an overview of the diagnostic workup, perioperative medical management, surgical approach, and surveillance strategy in patients with these challenging tumors.

Probability of positive genetic testing in patients diagnosed with pheochromocytoma and paraganglioma: Criteria beyond a family history.

BACKGROUND: Genetic testing for germline pheochromocytoma and paraganglioma susceptibility genes is associated with improved patient management. However, data are currently sparse on the probability of a positive testing result based on an individual's clinical presentation. This study evaluates clinical characteristics for association with testing positive for known pheochromocytoma and paraganglioma susceptibility genes. METHODS: This retrospective analysis examined 111 patients with a diagnosis of pheochromocytoma and paraganglioma who underwent genetic testing. Logistic regression and receiver operating characteristic analyses were performed to identify factors associated with a positive genetic testing result. Probabilities were then calculated for combinations of significant factors to determine the likelihood of a positive test result in each group. RESULTS: Of 32 patients with a family history of pheochromocytoma and paraganglioma, 31 (97%) had a germline mutation detected. Of 79 patients without a family history, 24 (30%) had a pathogenic germline mutation detected. In multivariate analysis, a positive family history, aged ≤47 years, and tumor size ≤2.9 cm were independent factors associated with a positive genetic testing result. Patients meeting all 3 criteria had a 100% probability compared with 13% in those without any of the criteria. In addition to a positive family history, having either aged ≤47 years or tumor size ≤2.9 cm resulted in a 90% and 100% probability of a positive result, respectively. In the absence of a family history, the probability in patients who were aged ≤47 years and had a tumor size ≤2.9 cm was 60%. CONCLUSION: In addition to a family history of pheochromocytoma and paraganglioma, aged ≤47 years, and tumor size ≤2.9 cm are associated with a higher probability of testing positive for a pheochromocytoma and paraganglioma susceptibility gene mutation. Patients meeting all 3 criteria have a 100% probability of a positive genetic testing result.

PAM staining intensity of primary neuroendocrine neoplasms is a potential prognostic biomarker.

Neuroendocrine neoplasms (NENs) are rare epithelial tumors with heterogeneous and frequently unpredictable clinical behavior. Available biomarkers are insufficient to guide individual patient prognosis or therapy selection. Peptidylglycine α-amidating monooxygenase (PAM) is an enzyme expressed by neuroendocrine cells that participates in hormone maturation. The objective of this study was to assess the distribution, clinical associations and survival implications of PAM immunoreactivity in primary NENs. Of 109 primary NENs, 7% were PAM-negative, 25% were PAM-low and 68% were PAM-high. Staining intensity was high in small bowel (p = 0.04) and low in stomach (p = 0.004) NENs. PAM staining was lower in higher grade tumors (p < 0.001) and patients who died (p < 0.001) but did not vary by tumor size or stage at surgery. In patients who died, time to death was shorter in patients with reduced PAM immunoreactivity: median times to death were 11.3 (PAM-negative), 29.4 (PAM-low) and 61.7 (PAM-high) months. Lower PAM staining was associated with increased risk of death after adjusting for disease stage [PAM negative, HR = 13.8 (CI: 4.2-45.5)]. PAM immunoreactivity in primary NENs is readily assessable and a potentially useful stage-independent predictor of survival.

Generation of highly potent DYRK1A-dependent inducers of human ß-Cell replication via Multi-Dimensional compound optimization.

Small molecule stimulation of ß-cell regeneration has emerged as a promising therapeutic strategy for diabetes. Although chemical inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is sufficient to enhance ß-cell replication, current lead compounds have inadequate cellular potency for in vivo application. Herein, we report the clinical stage anti-cancer kinase inhibitor OTS167 as a structurally novel, remarkably potent DYRK1A inhibitor and inducer of human ß-cell replication. Unfortunately, OTS167's target promiscuity and cytotoxicity curtails utility. To tailor kinase selectivity towards DYRK1A and reduce cytotoxicity we designed a library of fifty-one OTS167 derivatives based upon a modeled structure of the DYRK1A-OTS167 complex. Indeed, derivative characterization yielded several leads with exceptional DYRK1A inhibition and human ß-cell replication promoting potencies but substantially reduced cytotoxicity. These compounds are the most potent human ß-cell replication-promoting compounds yet described and exemplify the potential to purposefully leverage off-target activities of advanced stage compounds for a desired application.

Zinc-Chelating Small Molecules Preferentially Accumulate and Function within Pancreatic ß Cells.

Diabetes is a hyperglycemic condition characterized by pancreatic ß-cell dysfunction and depletion. Whereas methods for monitoring ß-cell function in vivo exist, methods to deliver therapeutics to ß cells are lacking. We leveraged the rare ability of ß cells to concentrate zinc to preferentially trap zinc-binding molecules within ß cells, resulting in ß-cell-targeted compound delivery. We determined that zinc-rich ß cells and islets preferentially accumulated TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) in a zinc-dependent manner compared with exocrine pancreas. Next, we asked whether appending a zinc-chelating moiety onto a ß-cell replication-inducing compound was sufficient to confer preferential ß-cell accumulation and activity. Indeed, the hybrid compound preferentially accumulated within rodent and human islets in a zinc-dependent manner and increased the selectivity of replication-promoting activity toward ß cells. These data resolve the fundamental question of whether intracellular accumulation of zinc-chelating compounds is influenced by zinc content. Furthermore, application of this principle yielded a proof-of-concept method for ß-cell-targeted drug delivery and bioactivity.

CC-401 Promotes ß-Cell Replication via Pleiotropic Consequences of DYRK1A/B Inhibition.

Pharmacologic expansion of endogenous ß cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control ß-cell growth we screened ∼2400 bioactive compounds for rat ß-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat ß-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) ß-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat ß cells, which were broadly growth responsive to compound treatment, human ß-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3ß (GSK-3ß) or activin A receptor type II-like kinase/transforming growth factor-ß (ALK5/TGF-ß) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human ß-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced ß-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the ß-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control ß-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control ß-cell growth.

Hyaluronan content governs tissue stiffness in pancreatic islet inflammation.

We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

Genetic Disruption of Adenosine Kinase in Mouse Pancreatic ß-Cells Protects Against High-Fat Diet-Induced Glucose Intolerance.

Islet ß-cells adapt to insulin resistance through increased insulin secretion and expansion. Type 2 diabetes typically occurs when prolonged insulin resistance exceeds the adaptive capacity of ß-cells. Our prior screening efforts led to the discovery that adenosine kinase (ADK) inhibitors stimulate ß-cell replication. Here, we evaluated whether ADK disruption in mouse ß-cells affects ß-cell mass and/or protects against high-fat diet (HFD)-induced glucose dysregulation. Mice targeted at the Adk locus were bred to Rip-Cre and Ins1-Cre/ERT1Lphi mice to enable constitutive (ßADKO) and conditional (ißADKO) disruption of ADK expression in ß-cells, respectively. Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion (GSIS) were longitudinally monitored in normal chow (NC)-fed and HFD-fed mice. In addition, ß-cell mass and replication were measured by immunofluorescence-based islet morphometry. NC-fed adult ßADKO and ißADKO mice displayed glucose tolerance, insulin tolerance and ß-cell mass comparable to control animals. By contrast, HFD-fed ßADKO and ißADKO animals had improved glucose tolerance and increased in vivo GSIS. Improved glucose handling was associated with increased ß-cell replication and mass. We conclude that ADK expression negatively regulates the adaptive ß-cell response to HFD challenge. Therefore, modulation of ADK activity is a potential strategy for enhancing the adaptive ß-cell response.

Electrically controlled release of insulin using polypyrrole nanoparticles.

Conducting polymers present an opportunity for developing programmable, adjustable, spatially, and temporally controllable drug delivery systems. While several small molecule drugs have been released from thin conductive polymeric films successfully, delivering large molecule therapeutics, such as polypeptides and nucleic acids, has remained a significant challenge. Poor drug loading (∼ng cm-2) of thin films coupled with film instability has, in many cases, made conducting polymer films refractory to clinical development. To address these limitations, we have utilized conductive polymer nanoparticulate backbones to controllably release insulin, a high molecular weight, clinically relevant polypeptide. We find that the interaction between insulin and the polymer scaffold can be described by a simple Langmuir-type adsorption model. By modifying the ratio of the amount of nanoparticles to the amount of insulin, we have obtained drug loading percentages estimated to be as high as 51 wt% percent. In vivo experiments in mice confirmed retained bioactivity of the released insulin after electrical stimulation.

A High-content In Vitro Pancreatic Islet ß-cell Replication Discovery Platform.

Loss of insulin-producing ß-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet ß-cells remains an attractive strategy. ß-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain ß-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify ß-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a ß-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on ß-cells and the molecular mechanisms that regulate ß-cell growth.