Insulinoma-derived pseudo-islets for diabetes research.

The islets of Langerhans of the pancreas are the primary endocrine organ responsible for regulating whole body glucose homeostasis. The use of isolated primary islets for research development and training requires organ resection, careful digestion, and isolation of the islets from nonendocrine tissue. This process is time consuming, expensive, and requires substantial expertise. For these reasons, we sought to develop a more rapidly obtainable and consistent model system with characteristic islet morphology and function that could be employed to train personnel and better inform experiments prior to using isolated rodent and human islets. Immortalized ß cell lines reflect several aspects of primary ß cells, but cell propagation in monolayer cell culture limits their usefulness in several areas of research, which depend on islet morphology and/or functional assessment. In this manuscript, we describe the propagation and characterization of insulinoma pseudo-islets (IPIs) from a rat insulinoma cell line INS832/3. IPIs were generated with an average diameter of 200 µm, consistent with general islet morphology. The rates of oxygen consumption and mitochondrial oxidation-reduction changes in response to glucose and metabolic modulators were similar to isolated rat islets. In addition, the dynamic insulin secretory patterns of IPIs were similar to primary rat islets. Thus, INS832/3-derived IPIs provide a valuable and convenient model for accelerating islet and diabetes research.

Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling.

BACKGROUND: All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. METHODS: Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of ß-cell function and RNA sequencing to elucidate transcriptomic changes. RESULTS: Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. CONCLUSIONS: These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved ß cell function.

Multivalent activation of GLP-1 and sulfonylurea receptors modulates ß-cell second-messenger signaling and insulin secretion.

Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) (GLP-1/Glb) for signaling efficacy in ß-cells. Expression of receptors for these ligands, as a combination, is relatively specific to the ß-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca2+, although Ca2+ responses were significantly depressed compared with the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at nonstimulatory glucose concentrations in INS 832/13 ß-cells or human islets of Langerhans. These data suggest that linking two binding elements, such as GLP-1 and Glb, into a single bivalent ligand can provide a unique functional agent targeted to ß-cells.

In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen demand, ß-cell function, and transcriptomes.

BACKGROUND: There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted. METHODS: Neonatal porcine islets (NPI; 1-3 days), juvenile porcine islets (JPI; 18-21 days), and adult porcine islets (API; 2+ years) were compared in vitro, including assessments of oxygen consumption rate, membrane integrity determined by FDA/PI staining, ß-cell proliferation, dynamic glucose-stimulated insulin secretion, and RNA sequencing. RESULTS: Oxygen consumption rate normalized to DNA was not significantly different between ages. Membrane integrity was age dependent, and API had the highest percentage of intact cells. API also had the highest glucose-stimulated insulin secretion response during a dynamic insulin secretion assay and had 50-fold higher total insulin content compared to NPI and JPI. NPI and JPI had similar glucose responsiveness, ß-cell percentage, and ß-cell proliferation rate. Transcriptome analysis was consistent with physiological assessments. API transcriptomes were enriched for cellular metabolic and insulin secretory pathways, while NPI exhibited higher expression of genes associated with proliferation. CONCLUSIONS: The oxygen demand, membrane integrity, ß-cell function and proliferation, and transcriptomes of islets from API, JPI, and NPI provide a comprehensive physiological comparison for future studies. These assessments will inform the optimal application of each age of porcine islet to expand the availability of islet transplantation.

Adrenergic receptor stimulation suppresses oxidative metabolism in isolated rat islets and Min6 cells.

Insulin secretion is stimulated by glucose metabolism and inhibited by catecholamines through adrenergic receptor stimulation. We determined whether catecholamines suppress oxidative metabolism in ß-cells through adrenergic receptors. In Min6 cells and isolated rat islets, epinephrine decreased oxygen consumption rates compared to vehicle control or co-administration of epinephrine with α2-adrenergic receptor antagonist yohimbine. Epinephrine also decreased forskolin-stimulated oxygen consumption rates, indicating cAMP dependent and independent actions. Furthermore, glucose oxidation rates were decreased with epinephrine, independent of the exocytosis of insulin, which was blocked with yohimbine. We evaluated metabolic targets through proteomic analysis after 4 h epinephrine exposure that revealed 466 differentially expressed proteins that were significantly enriched for processes including oxidative metabolism, protein turnover, exocytosis, and cell proliferation. These results demonstrate that acute α2-adrenergic stimulation suppresses glucose oxidation in ß-cells independent of nutrient availability and insulin exocytosis, while cAMP concentrations are elevated. Proteomics and immunoblots revealed changes in electron transport chain proteins that were correlated with lower metabolic reducing equivalents, intracellular ATP concentrations, and altered mitochondrial membrane potential implicating a new role for adrenergic control of mitochondrial function and ultimately insulin secretion.

Sorcin ablation plus ß-adrenergic stimulation generate an arrhythmogenic substrate in mouse ventricular myocytes.

Sorcin, a penta-EF hand Ca2+-binding protein expressed in cardiomyocytes, is known to interact with ryanodine receptors and other Ca2+ regulatory proteins. To investigate sorcin's influence on cardiac excitation-contraction coupling and its role in the development of cardiac malfunctions, we generated a sorcin knockout (KO) mouse model. Sorcin KO mice presented ventricular arrhythmia and sudden death when challenged by acute stress induced by isoproterenol plus caffeine. Chronic stress, which was induced by transverse aortic constriction, significantly decreased the survival rate of sorcin KO mice. Under isoproterenol stimulation, spontaneous Ca2+ release events were frequently observed in sorcin KO cardiomyocytes. Sorcin KO hearts of adult, but not young mice developed overexpression of L-type Ca2+ channel and Na+-Ca2+ exchanger, which enhanced ICa and INCX. Consequently, spontaneous Ca2+ release events in sorcin KO cardiomyocytes were more likely to induce arrhythmogenic delayed afterdepolarizations. Our study demonstrates sorcin deficiency may trigger cardiac ventricular arrhythmias due to Ca2+ disturbances, and evidences the critical role of sorcin in maintaining Ca2+ homeostasis, especially during the adrenergic response of the heart.

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

BACKGROUND: Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived ß cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high ß cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. METHODS: Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. RESULTS: Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic ß cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. CONCLUSIONS: In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to ß cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and ß cell function and leads to increased inflammatory signaling.

Linear scaffolds for multivalent targeting of melanocortin receptors.

Molecules bearing one, two, three, or four copies of the tetrapeptide His-dPhe-Arg-Trp were attached to scaffolds based on ethylene glycol, glycerol, and d-mannitol by means of the copper-assisted azide-alkyne cyclization. The abilities of these compounds to block binding of a probe at the melanocortin 4 receptor were evaluated using a competitive binding assay. All of the multivalent molecules studied exhibited 30- to 40-fold higher apparent affinites when compared to a monovalent control. These results are consistent with divalent binding to receptor dimers. No evidence for tri- or tetravalent binding was obtained. Differences in the interligand spacing required for divalent binding, as opposed to tri- or tetravalent binding, may be responsible for these results.

Synthesis and bioactivity of MSH4 oligomers prepared by an A2 + B2 strategy.

Oligomers incorporating the tetrapeptide MSH4, the minimum active sequence of melanocyte stimulating hormone, were synthesized by an A2 + B2 strategy involving microwave-assisted copper-catalyzed azide-alkyne cycloaddition. A2 contained an MSH4 core while B2 contained a (Pro-Gly)3 spacer. Soluble mixtures containing compounds with up to eight MSH4 units were obtained from oligomerizations at high monomer concentrations. The avidities of several oligomeric mixtures were evaluated by means of a competitive binding assay using HEK293 cells engineered to overexpress the melanocortin 4 receptor. When based on total MSH4 concentrations, avidities were only minimally enhanced compared with a monovalent control. The lack of variation in the effect of ligands on probe binding is consistent with high off rates for MSH4 in both monovalent and oligomeric constructs relative to that of the competing probe.

Development of a time-resolved fluorescence probe for evaluation of competitive binding to the cholecystokinin 2 receptor.

The synthesis, characterization, and use of Eu-DTPA-PEGO-Trp-Nle-Asp-Phe-NH2 (Eu-DTPA-PEGO-CCK4), a luminescent probe targeted to cholecystokinin 2 receptor (CCK2R, aka CCKBR), are described. The probe was prepared by solid phase synthesis. A Kd value of 17±2nM was determined by means of saturation binding assays using HEK-293 cells that overexpress CCK2R. The probe was then used in competitive binding assays against Ac-CCK4 and three new trivalent CCK4 compounds. Repeatable and reproducible binding assay results were obtained. Given its ease of synthesis, purification, receptor binding properties, and utility in competitive binding assays, Eu-DTPA-PEGO-CCK4 could become a standard tool for high-throughput screening of compounds in development targeted to cholecystokinin receptors.

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Trigonal scaffolds for multivalent targeting of melanocortin receptors.

Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4) were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane by means of the copper-assisted azide-alkyne cyclization. This synthetic design allows rapid assembly of multivalent molecules. The bioactivities of these compounds were evaluated using a competitive binding assay that employed human embryonic kidney cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (∼2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed.

Hetero-bivalent GLP-1/glibenclamide for targeting pancreatic ß-cells.

G protein-coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell-specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin-secreting pancreatic ß-cells are the sulfonylurea-1 (SUR1) and the glucagon-like peptide-1 (GLP-1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP-1 (7-36 GLP-1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to ß-cells, by using fluorescence microscopy or time-resolved saturation and competition binding assays, respectively. Once the ligand binds to ß-cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium-labelled GLP-1, likely a result of cooperative binding to the complementary receptors on the ßTC3 cells. The high-affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for ß-cell targeting in vivo.

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.

Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization.

As the endoplasmic reticulum (ER) is the compartment where disulfide bridges in secreted and cell surface proteins are formed, the disturbance of its redox state has profound consequences, yet regulation of ER redox potential remains poorly understood. To monitor the ER redox state in live cells, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. One particular green fluorescent protein (GFP)-based redox sensor, roGFP1-iL, could detect oxidizing changes in the ER despite having a reduction potential significantly lower than that previously reported for the ER. We have confirmed these observations and determined the mechanisms by which roGFP1-iL detects oxidizing changes. First, glutathione mediates the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitates dimerization of roGFP1-iL, which shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule's reduction potential compared with that of a dithiol redox buffer. We conclude that the glutathione redox couple in the ER significantly increased the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in the ER with a high degree of reliability in real time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggests a range of ER redox potentials consistent with those determined by earlier approaches that did not involve fluorescent sensors.

Multivalent Interactions: Synthesis and Evaluation of Melanotropin Multimers - Tools for Melanoma Targeting.

In order to develop agents for early detection and selective treatment of melanomas, high affinity and high specificity molecular tools are required. Enhanced specificity may be obtained by simultaneously binding to multiple cell surface targets via the use of multimeric analogs of naturally occurring ligands. Trimers targeting overexpressed melanocortin receptors have been found to be potential candidates for this purpose. In the present letter, we describe the synthesis and study of multimers based on a dendrimer-like scaffold. The binding affinity and activity results revealed that dendrimers promote multivalent interactions via statistical and/or cooperative effects on binding. Moreover, viability studies showed no significant toxicity at micromolar concentrations, which will allow these molecular complexes to be used in vivo. Finally, imaging studies showed effective internalization for all the molecules confirming their potential as delivery agents.

Pharmacogenomic strain differences in cardiovascular sensitivity to propofol.

INTRODUCTION: A pharmacogenomic approach was used to further localize the genetic region responsible for previously observed enhanced cardiovascular sensitivity to propofol in Dahl Salt Sensitive (SS) versus control Brown Norway (BN) rats. METHODS: Propofol infusion levels that decreased blood pressure by 50% were measured in BN.13(SS) rats (substitution of SS chromosome 13 into BN) and in five congenic (partial substitution) strains of SS.13(BN). The effect of superfused 2,6 diisopropylphenol on small mesenteric arterial vascular smooth muscle transmembrane potential was measured in congenic strains before and during superfusion with Rp-adenosine-3',5'-cyclic monophosphorothioate and 2.5 µM (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, inhibitors of protein kinase A and G, respectively. The genetic locus and potential role of the renin gene in mediating vascular smooth muscle sensitivity to propofol were determined in three selected subcongenic SS.BN¹³ strains. RESULTS: A 30-32% smaller propofol infusion rate reduced blood pressure by 50% in BN.13(SS) compared with BN and the SS.13(BN) congenic containing an 80 BN gene substitution. Compared with the 80 BN gene-containing SS.13(BN) congenic, SS exhibited greater protein kinase A dependent vascular smooth muscle hyperpolarization in response to propofol. Using subcongenics, the increased propofol-induced cardiovascular sensitivity and hyperpolarization was further localized to an eight-gene region (containing the BN renin gene). Blockade of angiotensin receptors with losartan in this subcongenic increased propofol-induced hyperpolarization by threefold to that observed in SS. CONCLUSIONS: Enhanced cardiovascular sensitivity to propofol in SS (compared with BN) is caused by an altered renin gene. Through modified second messenger function, this differentially regulates vascular smooth muscle contractile state and reduces vascular tone, thereby exacerbating cardiovascular depression by propofol.

Calcium homeostasis in human melanocytes: role of transient receptor potential melastatin 1 (TRPM1) and its regulation by ultraviolet light.

Transient receptor potential melastatin (TRPM) is a subfamily of ion channels that are involved in sensing taste, ambient temperature, low pH, osmolarity, and chemical ligands. Melastatin 1/TRPM1, the founding member, was originally identified as melanoma metastasis suppressor based on its expression in normal pigment cells in the skin and the eye but not in aggressive, metastasis-competent melanomas. The role of TRPM1 and its regulation in normal melanocytes and in melanoma progression is not understood. Here, we studied the relationship of TRPM1 expression to growth and differentiation of human epidermal melanocytes. TRPM1 expression and intracellular Ca(2+) levels are significantly lower in rapidly proliferating melanocytes compared to the slow growing, differentiated melanocytes. We show that lentiviral short hairpin RNA (shRNA)-mediated knockdown of TRPM1 results in reduced intracellular Ca(2+) and decreased Ca(2+) uptake suggesting a role for TRPM1 in Ca(2+) homeostasis in melanocytes. TRPM1 knockdown also resulted in a decrease in tyrosinase activity and intracellular melanin pigment. Expression of the tumor suppressor p53 by transfection or induction of endogenous p53 by ultraviolet B radiation caused repression of TRPM1 expression accompanied by decrease in mobilization of intracellular Ca(2+) and uptake of extracellular Ca(2+). These data suggest a role for TRPM1-mediated Ca(2+) homeostasis, which is also regulated by ultraviolet B, in melanogenesis.