scholarly journals Accelerating cryoprotectant diffusion kinetics improves cryopreservation of pancreatic islets

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nikola Dolezalova ◽  
Anja Gruszczyk ◽  
Kerry Barkan ◽  
John A. Gamble ◽  
Sam Galvin ◽  
...  
Keyword(s):  
Small Molecules ◽  
Pancreatic Islets ◽  
Incubation Temperature ◽  
Diabetic Patients ◽  
Diffusion Kinetics ◽  
Β Cells ◽  
Multicellular Aggregates ◽  
Mouse Islets ◽  
Peptide Secretion ◽  
Cryopreservation Method

AbstractCryopreservation offers the potential to increase the availability of pancreatic islets for treatment of diabetic patients. However, current protocols, which use dimethyl sulfoxide (DMSO), lead to poor cryosurvival of islets. We demonstrate that equilibration of mouse islets with small molecules in aqueous solutions can be accelerated from > 24 to 6 h by increasing incubation temperature to 37 °C. We utilize this finding to demonstrate that current viability staining protocols are inaccurate and to develop a novel cryopreservation method combining DMSO with trehalose pre-incubation to achieve improved cryosurvival. This protocol resulted in improved ATP/ADP ratios and peptide secretion from β-cells, preserved cAMP response, and a gene expression profile consistent with improved cryoprotection. Our findings have potential to increase the availability of islets for transplantation and to inform the design of cryopreservation protocols for other multicellular aggregates, including organoids and bioengineered tissues.

scholarly journals TLR4 signals through islet macrophages to alter cytokine secretion during diabetes

2020 ◽  
Vol 247 (1) ◽  
pp. 87
Author(s):  
Manesh Chittezhath ◽  
Cho M M Wai ◽  
Vanessa S Y Tay ◽  
Minni Chua ◽  
Sarah R Langley ◽  
...  
Keyword(s):  
Pancreatic Islets ◽  
Expression Patterns ◽  
Hormone Secretion ◽  
Specific Cell ◽  
Diabetic Mice ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Β Cells ◽  
Mouse Islets ◽  
Tlr Signalling

Toll-like receptors (TLRs), particularly TLR4, may act as immune sensors for metabolic stress signals such as lipids and link tissue metabolic changes to innate immunity. TLR signalling is not only tissue-dependent but also cell-type dependent and recent studies suggest that TLRs are not restricted to innate immune cells alone. Pancreatic islets, a hub of metabolic hormones and cytokines, respond to TLR signalling. However, the source of TLR signalling within the islet remain poorly understood. Uncovering the specific cell source and its role in mediating TLR signalling, especially within type 2 diabetes (T2D) islet will yield new targets to tackle islet inflammation, hormone secretion dysregulation and ultimately diabetes. In the present study, we immuno-characterised TLRs linked to pancreatic islets in both healthy and obese diabetic mice. We found that while TLRs1–4 and TLR9 were expressed in mouse islets, these TLRs did not co-localise with insulin-producing β-cells. β-Cells from obese diabetic mice were also devoid of these TLRs. While TLR immunoreactivity in obese mice islets increased, this was driven mostly by increased islet endothelial cell and islet macrophage presence. Analysis of human islet single-cell RNA-seq databases revealed that macrophages were an important source of islet TLRs. However, only TLR4 and TLR8 showed variation and cell-type specificity in their expression patterns. Cell depletion experiments in isolated mouse islets showed that TLR4 signalled through macrophages to alter islet cytokine secretome. Together, these studies suggest that islet macrophages are a dominant source of TLR4-mediated signalling in both healthy and diabetic islets.

scholarly journals Glucose stimulates microRNA-199 expression in murine pancreatic β-cells

2019 ◽  
Vol 295 (5) ◽  
pp. 1261-1270 ◽  
Author(s):  
Joao Pedro Werneck-de-Castro ◽  
Manuel Blandino-Rosano ◽  
Denise Hilfiker-Kleiner ◽  
Ernesto Bernal-Mizrachi
Keyword(s):  
Promoter Activity ◽  
Transcriptional Activity ◽  
Cell Function ◽  
Calcium Influx ◽  
Diabetic Patients ◽  
Mrna Levels ◽  
Β Cells ◽  
Min6 Cells ◽  
Mouse Islets ◽  
Insulinoma Cells

MicroRNA 199 (miR-199) negatively impacts pancreatic β-cell function and its expression is highly increased in islets from diabetic mice as well as in plasma of diabetic patients. Here we investigated how miR-199 expression is regulated in β-cells by assessing expression of miR-199 precursors (primiR-199a1, primiR-199a2, and primiR-199b) and mature miR-199 (miR-199-3p and miR-199-5p) and promoter transcriptional activity assays in mouse islets and mouse insulinoma cells (MIN6) under different stimuli. We found that mouse islets equally express miR-199-3p and miR-199-5p. However, the primiRNA expression levels differed; although primiR-199a1 expression was about 30% greater than that of primiR-199a2, primiR-199b is barely detected in islets. We observed a 2-fold increase in primiR-199a1 and primiR-199a2 mRNA levels in mouse islets cultured in 10 mm glucose compared with 5.5 mm glucose. Similar responses to glucose were observed in MIN6 cells. Exposure to 30 mm KCl to induce membrane depolarization and calcium influx increased expression of primiR-199a2 but not of primiR-199a1 in MIN6 cells, indicating that calcium influx was involved. Transcriptional activity studies in MIN6 cells also revealed that primiR-199a2 promoter activity was enhanced by glucose and reduced by 2-deoxy-D-glucose–induced starvation. KCl and the potassium channel blocker tolbutamide also stimulated primiR-199a2 promoter activity. Calcium channel blockade by nifedipine reduced primiR-199a2 promoter activity in MIN6 cells, and diazoxide-mediated calcium influx inhibition blunted glucose up-regulation of miR-199-3p in islets. In conclusion, we uncover that glucose acutely up-regulates miR-199 family expression in β-cells. Glucose metabolism and calcium influx are involved in primiR-199a2 expression but not primiR-199a1 expression.

scholarly journals Is there a role for the RNA-binding protein LARP1 in β-cells?

2020 ◽  
Author(s):  
Joao Pedro Werneck-de-Castro ◽  
Flavia Leticia Martins Peçanha ◽  
Diego Silvestre ◽  
Ernesto Bernal-Mizrachi
Keyword(s):  
Weight Gain ◽  
Amino Acid ◽  
Glucose Tolerance ◽  
Glucose Homeostasis ◽  
Protein Translation ◽  
Diabetic Patients ◽  
Β Cells ◽  
Branched Chain Amino Acid ◽  
Mouse Islets ◽  
Branched Chain

ABSTRACTMechanistic target of rapamycin complex 1 (mTORC1) is a cellular rheostat linking nutrient availability and growth factor to cellular protein translation. In pancreatic insulin secreting β-cells, mTORC1 deficiency or chronic hyperactivation leads to diabetes. mTORC1 complexes with La-related protein 1 (LARP1) to specifically regulate the expression of 5’ terminal oligopyrimidine tract (5’TOP) mRNAs which encode proteins of the translation machinery and ribosome biogenesis. We aimed to investigate the role played by LARP1 in β-cells in vivo. Here we show that LARP1 is the most expressed LARP in mouse islets and human β-cells, being 2-4-fold more abundant than LARP1B, a member of the family that also interacts with mTORC1. Interestingly, β-cells from diabetic patients have higher LARP1 and LARP1B expression suggesting greater protein translation. These studies led us to generate a conditional LARP1 knockout mouse in β-cells (β-Larp1KO mice). These mice exhibit normal levels of all LARP family members including Larp1B, Larp4, Larp6 and Larp7. We did not observe any difference between control and β-Larp1KO male mice in body weight gain, glucose levels and glucose tolerance at 8, 14 and 44 weeks of age. Female β-Larp1KO mice also performed normally during the glucose tolerance test. We then challenged the β-Larp1KO mice with high fat (HFD) or high branched-chain amino acid (BCAA) diets. During the course of 8 weeks in HFD, β-Larp1KO and control mice had similar weight gain and did not show alterations in glucose homeostasis compared to control littermates. BCAA did not impair glucose metabolism up to 8 weeks of diet challenge. However, glucose tolerance was slightly impaired in the β-Larp1KO mice at 16 weeks under BCAA diet. In conclusion, LARP1 is the most abundant LARP in mouse islets and human β-cells and it is upregulated in diabetic subjects. While the lack of LARP1 specifically in β-cells did not alter glucose homeostasis in basal conditions, long-term high branched-chain amino acid feeding could impair glucose tolerance.

scholarly journals The RNA-binding protein LARP1 is dispensable for pancreatic β-cell function and mass

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joao Pedro Werneck-de-Castro ◽  
Flavia Leticia Martins Peçanha ◽  
Diego Henrique Silvestre ◽  
Ernesto Bernal-Mizrachi
Keyword(s):  
Glucose Homeostasis ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Cell Function ◽  
Cell Mass ◽  
Diabetic Patients ◽  
Β Cells ◽  
Β Cell ◽  
Fasting Glycemia ◽  
Mouse Islets

AbstractMechanistic target of rapamycin complex 1 (mTORC1) deficiency or chronic hyperactivation in pancreatic β-cells leads to diabetes. mTORC1 complexes with La-related protein 1 (LARP1) to specifically regulate the expression of 5′ terminal oligopyrimidine tract (5′TOP) mRNAs which encode proteins of the translation machinery and ribosome biogenesis. Here we show that LARP1 is the most expressed LARP in mouse islets and human β-cells, being 2–4-fold more abundant than LARP1B, a member of the family that also interacts with mTORC1. Interestingly, β-cells from diabetic patients have higher LARP1 and LARP1B expression. However, specific deletion of Larp1 gene in β-cells (β-Larp1KO mice) did not impair insulin secretion and glucose metabolism in male and female mice. High fat or high branched-chain amino acid (BCAA) diets did not disturb glucose homeostasis compared to control littermates up to 8 weeks; BCAA diet slightly impaired glucose tolerance in the β-Larp1KO mice at 16 weeks. However, no differences in plasma insulin levels, non-fasting glycemia and β-cell mass were observed in the β-Larp1KO mice. In conclusion, LARP1 is the most abundant LARP in mouse islets and human β-cells, and it is upregulated in diabetic subjects. However, genetically disruption of Larp1 gene did not impact glucose homeostasis in basal and diabetogenic conditions, suggesting no major role for LARP1 in β-cells.

IONIC EFFECTS ON THE UPTAKE OF CHLOROMERCURIBENZENE-p-SULPHONIC ACID BY PANCREATIC ISLETS

1977 ◽  
Vol 86 (3) ◽  
pp. 552-560 ◽  
Author(s):  
Monica Söderberg ◽  
Inge-Bert Täljedal
Keyword(s):  
Pancreatic Islets ◽  
Islet Cell ◽  
Choline Chloride ◽  
Inorganic Ions ◽  
Sulphonic Acid ◽  
Cell Uptake ◽  
Β Cells ◽  
Sulphydryl Groups ◽  
Microdissected Pancreatic Islets ◽  
Ionic Effects

ABSTRACT Effects of inorganic ions on the uptake of chloromercuribenzene-p-sulphonic acid (CMBS) were studied in microdissected pancreatic islets of non-inbred ob/ob-mice. Na2SO4 stimulated the total islet cell uptake of CMBS but decreased the amount of CMBS remaining in islets after brief washing with L-cysteine. CaCl2 stimulated both the total and the cysteine-non-displaceable uptake; the stimulatory effect of CaCl2 on the cysteine-non-displaceable CMBS uptake was counteracted by Na2SO4. NaCl, KCl or choline chloride had no significant effect on the total islet cell uptake of CMBS, whereas LiCl was stimulatory. It is concluded that β-cells resemble erythrocytes in having a permeation path for CMBS that is inhibited by SO42−. By analogy with existing models of the erythrocyte membrane, it is suggested that the SO42−-sensitive path leads to sulphydryl groups controlling monovalent cationic permeability in β-cells.

Influence of residual C-peptide secretion on nocturnal serum TSH peak in well-controlled diabetic patients

1997 ◽  
Vol 47 (3) ◽  
pp. 305-310 ◽  
Author(s):  
V. Coiro ◽  
R. Volpi ◽  
C. Marchesi ◽  
L. Capretti ◽  
G. Speroni ◽  
...  
Keyword(s):  
Diabetic Patients ◽  
C Peptide ◽  
Peptide Secretion ◽  
Serum Tsh

scholarly journals Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

2010 ◽  
Vol 298 (4) ◽  
pp. E807-E814 ◽  
Author(s):  
Lara R. Nyman ◽  
Eric Ford ◽  
Alvin C. Powers ◽  
David W. Piston
Keyword(s):  
Blood Flow ◽  
Blood Glucose ◽  
Pancreatic Islets ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Types ◽  
Β Cells ◽  
Islet Blood Flow ◽  
Significant Difference

Pancreatic islets are highly vascularized and arranged so that regions containing β-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic (∼300 mg/dl or 16.8 mM) and hypoglycemic (∼50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the β-cells or yellow fluorescent protein in the α-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of β-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

scholarly journals Zinc-Chelating Small Molecules Preferentially Accumulate and Function within Pancreatic β Cells

2019 ◽  
Vol 26 (2) ◽  
pp. 213-222.e6 ◽  
Author(s):  
Timothy M. Horton ◽  
Paul A. Allegretti ◽  
Sooyeon Lee ◽  
Hannah P. Moeller ◽  
Mark Smith ◽  
...  
Keyword(s):  
Small Molecules ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
And Function

Effects of pharmacological inhibition of NADPH oxidase or iNOS on pro-inflammatory cytokine, palmitic acid or H2O2-induced mouse islet or clonal pancreatic β-cell dysfunction

2010 ◽  
Vol 30 (6) ◽  
pp. 445-453 ◽  
Author(s):  
Marta Michalska ◽  
Gabriele Wolf ◽  
Reinhard Walther ◽  
Philip Newsholme
Keyword(s):  
Fatty Acids ◽  
Insulin Secretion ◽  
Nadph Oxidase ◽  
Inflammatory Cytokine ◽  
Pancreatic Β Cell ◽  
Β Cells ◽  
Β Cell ◽  
Cell Dysfunction ◽  
Mouse Islets ◽  
Pro Inflammatory Cytokines

Various pancreatic β-cell stressors including cytokines and saturated fatty acids are known to induce oxidative stress, which results in metabolic disturbances and a reduction in insulin secretion. However, the key mechanisms underlying dysfunction are unknown. We investigated the effects of prolonged exposure (24 h) to pro-inflammatory cytokines, H2O2 or PA (palmitic acid) on β-cell insulin secretion, ATP, the NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) component p47phox and iNOS (inducible nitric oxide synthase) levels using primary mouse islets or clonal rat BRIN-BD11 β-cells. Addition of a pro-inflammatory cytokine mixture [IL-1β (interleukin-1β), TNF-α (tumour necrosis factor-α) and IFN-γ (interferon-γ)] or H2O2 (at sub-lethal concentrations) inhibited chronic (24 h) levels of insulin release by at least 50% (from islets and BRIN-BD11 cells), while addition of the saturated fatty acid palmitate inhibited acute (20 min) stimulated levels of insulin release from mouse islets. H2O2 decreased ATP levels in the cell line, but elevated p47phox and iNOS levels as did cytokine addition. Similar effects were observed in mouse islets with respect to elevation of p47phox and iNOS levels. Addition of antioxidants SOD (superoxide dismutase), Cat (catalase) and NAC (N-acetylcysteine) attenuated H2O2 or the saturated fatty acid palmitate-dependent effects, but not cytokine-induced dysfunction. However, specific chemical inhibitors of NADPH oxidase and/or iNOS appear to significantly attenuate the effects of cytokines, H2O2 or fatty acids in islets. While pro-inflammatory cytokines are known to increase p47phox and iNOS levels in β-cells, we now report that H2O2 can increase levels of the latter two proteins, suggesting a key role for positive-feedback redox sensitive regulation of β-cell dysfunction.

scholarly journals Regulation of glucokinase in pancreatic islets by prolactin: a mechanism for increasing glucose-stimulated insulin secretion during pregnancy

2007 ◽  
Vol 193 (3) ◽  
pp. 367-381 ◽  
Author(s):  
Anthony J Weinhaus ◽  
Laurence E Stout ◽  
Nicholas V Bhagroo ◽  
T Clark Brelje ◽  
Robert L Sorenson
Keyword(s):  
Insulin Secretion ◽  
Glucose Metabolism ◽  
Pancreatic Islets ◽  
Glucose Transporter ◽  
Β Cells ◽  
Glucose Transporter 2 ◽  
Underlying Mechanisms ◽  
Glucose Stimulated Insulin Secretion ◽  
Induced Changes

Glucokinase activity is increased in pancreatic islets during pregnancy and in vitro by prolactin (PRL). The underlying mechanisms that lead to increased glucokinase have not been resolved. Since glucose itself regulates glucokinase activity in β-cells, it was unclear whether the lactogen effects are direct or occur through changes in glucose metabolism. To clarify the roles of glucose metabolism in this process, we examined the interactions between glucose and PRL on glucose metabolism, insulin secretion, and glucokinase expression in insulin 1 (INS-1) cells and rat islets. Although the PRL-induced changes were more pronounced after culture at higher glucose concentrations, an increase in glucose metabolism, insulin secretion, and glucokinase expression occurred even in the absence of glucose. The presence of comparable levels of insulin secretion at similar rates of glucose metabolism from both control and PRL-treated INS-1 cells suggests the PRL-induced increase in glucose metabolism is responsible for the increase in insulin secretion. Similarly, increases in other known PRL responsive genes (e.g. the PRL receptor, glucose transporter-2, and insulin) were also detected after culture without glucose. We show that the upstream glucokinase promoter contains multiple STAT5 binding sequences with increased binding in response to PRL. Corresponding increases in glucokinase mRNA and protein synthesis were also detected. This suggests the PRL-induced increase in glucokinase mRNA and its translation are sufficient to account for the elevated glucokinase activity in β-cells with lactogens. Importantly, the increase in islet glucokinase observed with PRL is in line with that observed in islets during pregnancy.

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