Functional and Immunohistochemical Evaluation of Porcine Neonatal Islet-Like Cell Clusters

2003 ◽  
Vol 12 (1) ◽  
pp. 13-25 ◽  
Author(s):  
T. B. Nielsen ◽  
K. B. Yderstraede ◽  
H. D. Schrøder ◽  
Jens Juul Holst ◽  
Klaus Brusgaard ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Nude Mice ◽  
Cell Mass ◽  
Diabetic Patients ◽  
Type I ◽  
Fourfold Increase ◽  
Cell Clusters ◽  
Glucose Challenge

Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transplantation to athymic nude mice. On average we obtained 29,000 NICCs from each pancreas. In a perifusion system, NICCs responded poorly to a glucose challenge alone, but 10 mmol/L arginine elicited a fourfold increase in insulin secretion and 16.7 mmol/L glucose + 10 mmol/L arginine caused a sevenfold increase in insulin secretion, indicating some sensitivity towards glucose. Hormone content as well as the number of hormone-containing cells increased for the first 14 days of culture. When NICCs were stained for hormones, proliferation (Ki67), and duct cells (CK7), some insulin- and glucagon-positive cells co-stained for proliferation. However no co-staining was observed between insulin- and glucagon-positive cells or between hormone- and CK7-positive cells. Following transplantation of 2000 NICCs under the renal capsule of diabetic nude mice, BG levels were normalized within an average of 13 weeks. Oral and IP glucose tolerance tests revealed a normal or even faster clearance of a glucose load compared with normal controls. Immunohistochemical examination of the grafts revealed primarily insulin-positive cells. In summary, in vitro, NICCs responded to a challenge including glucose and arginine. There was a potential for expansion of the β-cell mass of NICCs in vitro as well as in vivo where NICCs eventually may normalize blood glucose of diabetic mice.

scholarly journals ECM-enriched alginate hydrogels for bioartificial pancreas: an ideal niche to improve insulin secretion and diabetic glucose profile

2019 ◽  
Vol 17 (4) ◽  
pp. 228080001984892 ◽  
Author(s):  
Joana Crisóstomo ◽  
Ana M Pereira ◽  
Sílvia J Bidarra ◽  
Ana C Gonçalves ◽  
Pedro L Granja ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Diabetic Patients ◽  
Bioartificial Pancreas ◽  
Multicellular Spheroids ◽  
Rgd Peptides ◽  
Alginate Hydrogels ◽  
And Function

Introduction: The success of a bioartificial pancreas crucially depends on ameliorating encapsulated beta cells survival and function. By mimicking the cellular in vivo niche, the aim of this study was to develop a novel model for beta cells encapsulation capable of establishing an appropriate microenvironment that supports interactions between cells and extracellular matrix (ECM) components. Methods: ECM components (Arg-Gly-Asp, abbreviated as RGD) were chemically incorporated in alginate hydrogels (alginate-RGD). After encapsulation, INS-1E beta cells outcome was analyzed in vitro and after their implantation in an animal model of diabetes. Results: Our alginate-RGD model demonstrated to be a good in vitro niche for supporting beta cells viability, proliferation, and activity, namely by improving the key feature of insulin secretion. RGD peptides promoted cell–matrix interactions, enhanced endogenous ECM components expression, and favored the assembly of individual cells into multicellular spheroids, an essential configuration for proper beta cell functioning. In vivo, our pivotal model for diabetes treatment exhibited an improved glycemic profile of type 2 diabetic rats, where insulin secreted from encapsulated cells was more efficiently used. Conclusions: We were able to successfully introduce a novel valuable function in an old ally in biomedical applications, the alginate. The proposed alginate-RGD model stands out as a promising approach to improve beta cells survival and function, increasing the success of this therapeutic strategy, which might greatly improve the quality of life of an increasing number of diabetic patients worldwide.

Nonenzymatic glycation of human platelet membrane proteins in vitro and in vivo.

1986 ◽  
Vol 32 (7) ◽  
pp. 1328-1331 ◽  
Author(s):  
T Sampietro ◽  
S Lenzi ◽  
P Cecchetti ◽  
O Giampietro ◽  
L Cruschelli ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Glucose Concentration ◽  
Time Course ◽  
Human Platelet ◽  
Platelet Membrane ◽  
Diabetic Patients ◽  
Type I ◽  
Nonenzymatic Glycation

Abstract Human platelet membrane proteins (PMP), incubated in vitro in the presence of various concentrations of glucose, undergo nonenzymatic glycation, as evidenced by incorporation of [3-3H]glucose radioactivity into the acid-precipitable fraction. The time course of the reaction is linear for the first hours, and the rate of glycation depends on the glucose concentration in the medium: at a glucose concentration of 80 mmol/L, up to 60 nmol of glucose is bound per milligram of PMP. The ketoaminic nature of the glucose/protein linkages was demonstrated by the finding of 5-hydroxymethylfurfuraldehyde by liquid-chromatographic analysis of acid hydrolysates of PMP. We analyzed PMP from 13 subjects with type I poorly controlled diabetes and from 10 nondiabetics. Nonenzymatic glycation, evaluated as nanomoles of the aldehyde per milligram of protein, was much greater in diabetic patients than in nondiabetics: 1.58 +/- 0.70 vs 0.37 +/- 0.18 (mean +/- SD).

scholarly journals AWRK6, a Novel GLP-1 Receptor Agonist, Attenuates Diabetes by Stimulating Insulin Secretion

2018 ◽  
Vol 19 (10) ◽  
pp. 3053
Author(s):  
Qiuyu Wang ◽  
Chunlin Zhao ◽  
Lili Jin ◽  
Hanyu Zhang ◽  
Qifan Miao ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Mass ◽  
Membrane Integrity ◽  
The Body ◽  
Oral Glucose ◽  
Diabetic Mice ◽  
Β Cell ◽  
Min6 Cells

Diabetes is a metabolic disorder leading to many complications. The treatment of diabetes mainly depends on hypoglycemic drugs, often with side effects, which drive us to develop novel agents. AWRK6 was a peptide developed from the antimicrobial peptide Dybowskin-2CDYa in our previous study, and the availability of AWRK6 on diabetes intervention was unknown. Here, in vivo and in vitro experiments were carried out to investigate the effects of AWRK6 against diabetes. In diabetic mice, induced by high-fat diet followed by streptozocin (STZ) administration, the daily administration of AWRK6 presented acute and sustained hypoglycemic effects. The plasma insulin was significantly elevated by AWRK6 during an oral glucose tolerance test (OGTT). The relative β cell mass in diabetic mice was increased by AWRK6 treatment. The body weight and food intake were remarkably reduced by AWRK6 administration. In the mouse pancreatic β cell line Min6 cells, the intracellular calcium concentration was found to be enhanced under the treatment with AWRK6, and protein kinase A (PKA) inhibitor H-89 and Epac2 inhibitor HJC0350 represented inhibitory effects of the insulinotropic function of AWRK6. By FITC-AWRK6 incubation and GLP-1 receptor (GLP-1R) knockdown, AWRK6 proved to be a novel GLP-1R agonist. In addition, AWRK6 showed no toxicity in cell viability and membrane integrity in Min6 cells, and no hypoglycemia risk and no lethal toxicity in mice. In summary, AWRK6 was found as a novel agonist of GLP-1R, which could stimulate insulin secretion to regulate blood glucose and energy metabolism, via cAMP-calcium signaling pathway, without significant toxicity. The peptide AWRK6 might become a novel candidate for diabetes treatment.

Accelerated Functional Maturation of Isolated Neonatal Porcine Cell Clusters: In Vitro and In Vivo Results in NOD Mice

2005 ◽  
Vol 14 (5) ◽  
pp. 249-261 ◽  
Author(s):  
Giovanni Luca ◽  
Claudio Nastruzzi ◽  
Mario Calvitti ◽  
Ennio Becchetti ◽  
Tiziano Baroni ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Time Lag ◽  
Cell Mass ◽  
Nod Mice ◽  
Β Cell ◽  
Cell Clusters ◽  
Porcine Islets ◽  
Neonatal Porcine Islets

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the β-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from <3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in β-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional β-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

scholarly journals Regulation of islet function and gene expression by prolactin during pregnancy

2019 ◽  
Author(s):  
Vipul Shrivastava ◽  
Megan Lee ◽  
Marle Pretorius ◽  
Guneet Makkar ◽  
Carol Huang
Keyword(s):  
Gene Expression ◽  
Insulin Secretion ◽  
Serum Insulin ◽  
Prolactin Receptor ◽  
Cell Mass ◽  
Insulin Level ◽  
Β Cells ◽  
Β Cell

AbstractPancreatic islets adapt to insulin resistance of pregnancy by up regulating β-cell proliferation and increase insulin secretion. Previously, we found that prolactin receptor (Prlr) signaling is important for this process, as heterozygous prolactin receptor-null (Prlr+/−) mice are glucose intolerant, had a lower number of β cells and lower serum insulin levels than wild type mice during pregnancy. However, since Prlr expression is ubiquitous, to determine its β-cell specific effects, we generated a transgenic mouse with a floxed Prlr allele under the control of an inducible promoter, allowing conditional deletion of Prlr from β cells in adult mice. In this study, we found that β-cell-specific Prlr reduction resulted in elevated blood glucose during pregnancy. Similar to our previous finding in mouse with global Prlr reduction, β-cell-specific Prlr loss led to a lower β-cell mass and a lower in vivo insulin level during pregnancy. However, these islets do not have an intrinsic insulin secretion defect when tested in vitro. Interestingly, when we compared the islet gene expression profile, using islets isolated from mice with global versus β-cell-specific Prlr reduction, we found some important differences in genes that regulate apoptosis and insulin secretion. This suggests that Prlr has both cell-autonomous and non-cell-autonomous effect on β cells, beyond its regulation of pro-proliferative genes.

In vitro and in vivo evaluation of insulin-producing βTC6-F7 cells in microcapsules

1998 ◽  
Vol 274 (5) ◽  
pp. C1356-C1362 ◽  
Author(s):  
D. Zhou ◽  
A. M. Sun ◽  
X. Li ◽  
S. N. Mamujee ◽  
I. Vacek ◽  
...  
Keyword(s):  
Cell Mass ◽  
Three Dimensional ◽  
In Vivo Studies ◽  
Oral Glucose ◽  
In Vivo Evaluation ◽  
Glucose Response ◽  
Encapsulated Cells ◽  
Glucose Challenge

In the present study, the insulin secretory capacity of βTC6-F7 cells in microcapsules was evaluated. The cell mass within capsules was found to expand in a three-dimensional fashion, in contrast to cells seeded on plates that grew as a monolayer. In in vitro studies, both free and encapsulated cells were found to secrete insulin in the absence of glucose, at 13.6 ± 1.1 and 14.5 ± 0.9 ng ⋅ 106cells−1 ⋅ 60 min−1, respectively, with the response rising to a maximum of 26.0 ± 0.8 and 31 ± 2.3 ng ⋅ 106cells−1 ⋅ 60 min−1 in the presence of 16.8 mM glucose. Encapsulated cells were able to produce Ca2+ responses in the presence of KCl (50 mM) and BAY K 8644 (100 μM). In in vivo studies, intraperitoneal transplantation of 3.0 ×106 microencapsulated cells into mice ( n = 5) with streptozotocin-induced diabetes resulted in the restoration of normoglycemia up to 57 days. Insulin concentrations rose from 0.4 ± 0.1 ng/ml before the graft administration to 2.2 ± 0.8 ng/ml after the transplantation in the normoglycemic recipients. An oral glucose challenge in transplant recipients demonstrated a flat glucose response, suggesting extremely high glucose clearance rates. These data demonstrate the potential use of the immunoisolated β-cell lines for the treatment of diabetes.

scholarly journals Isoform-specific roles of prolyl hydroxylases in the regulation of pancreatic β-cell function

Endocrinology ◽  
2021 ◽  
Author(s):  
Monica Hoang ◽  
Emelien Jentz ◽  
Sarah M Janssen ◽  
Daniela Nasteska ◽  
Federica Cuozzo ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Katp Channel ◽  
Cell Mass ◽  
Isolated Islets ◽  
Second Phase ◽  
Β Cells ◽  
Β Cell ◽  
Prolyl Hydroxylases ◽  
Glucose Challenge

Abstract Pancreatic β-cells can secrete insulin via two pathways characterized as KATP channel-dependent and independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP + ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α (HIFα). In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell specific knockout (KO) mice for all three isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the 1 st phase of insulin secretion but enhanced the second-phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP + ratio. All three PHD isoforms are expressed in β-cells, with PHD3 showing the most unique expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Calibration of a Wearable Glucose Sensor

1992 ◽  
Vol 15 (1) ◽  
pp. 55-61 ◽  
Author(s):  
F.J. Schmidt ◽  
A.L. Aalders ◽  
A.J.M. Schoonen ◽  
H. Doorenbos
Keyword(s):  
Blood Glucose ◽  
Healthy Volunteers ◽  
Glucose Oxidase ◽  
Glucose Sensor ◽  
Subcutaneous Tissue ◽  
The Body ◽  
Diabetic Patients ◽  
Type I

Calibration of glucose sensors proved difficult for electrodes with immobilized glucose-oxidase. The correlation between the sensitivity of the electrodes in vitro and in vivo appeared to be poor. We developed a new type of glucose sensor, based on a microdialysis system, in which an oxygen electrode is used as detector outside the body and the enzyme glucose-oxidase dissolved in water is used as a dynamic selector. The enzyme solution is pumped through a hollow fiber placed subcutaneously, before the fluid passes the detector. The glucose sensor was tested in the subcutaneous abdominal tissue of 12 healthy volunteers and 12 type I diabetic patients. Blood glucose was clamped at two levels to permit a two-point calibration of the sensor in vivo. These values correlated well with the in vitro calibration factors (r=0.949). In subcutaneous tissue the sensor measures 43 ± 9% of the blood glucose value, using the in vitro calibration factor. No differences were detected between healthy volunteers and diabetic patients.

Comparison of Two Methods of Pancreas Islets Immunoisolation

1997 ◽  
Vol 20 (12) ◽  
pp. 701-703 ◽  
Author(s):  
T. Orłowski ◽  
E. Sitarek ◽  
K. Tatarkiewicz ◽  
M. Sabat ◽  
M. Antosiak
Keyword(s):  
Insulin Secretion ◽  
Blood Glucose ◽  
Fasting Blood Glucose ◽  
Diabetic Mice ◽  
In Vitro Methods ◽  
Glucose Challenge ◽  
In Vitro Insulin Secretion ◽  
Islets Transplantation

The efficacy of two methods of Langerhans islets immunoisolation was compared. For this purpose the function of islets encapsulated with alginate/polyethylenimine/protamine/heparin (APPH) or with alginate/poly-L-lisine/alginate (APA) membranes was assessed: in vitro according to their survival and response to glucose challenges, and in vivo according to their capability to provide sufficient insulin delivery to maintain normal fasting blood glucose following xenotransplantation to streptozotocin diabetic mice. In vitro insulin secretion and the response to glucose challenge of APPH and APA encapsulated islets were comparable to free islets. In vivo intraperitoneal concordant xenotransplantation of APA encapsulated rat islets reversed the diabetic state of streptozotocin diabetic mice for a longer period, than APPH islet grafts. This study clearly demonstrated the inadequacy of in vitro methods in the prediction of in vivo results of islets transplantation.

scholarly journals Small Islets Transplantation Superiority to Large Ones: Implications from Islet Microcirculation and Revascularization

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Wenjuan Li ◽  
Ruxing Zhao ◽  
Jidong Liu ◽  
Meng Tian ◽  
Yiran Lu ◽  
...  
Keyword(s):  
Cell Mass ◽  
Diabetic Patients ◽  
Vascular Endothelial ◽  
Pancreatic Islet Transplantation ◽  
Histological Phenotype ◽  
And Function ◽  
Endothelial Growth Factor ◽  
Selection Of

Pancreatic islet transplantation is a promising therapy to regain glycemic control in diabetic patients. The selection of ideal grafts is the basis to guarantee short-term effectivity and longevity of the transplanted islets. Contradictory to the traditional notion, recent findings implied the superiority of small islets for better transplantation outcomes rather than the large and intact ones. However, the mechanisms remain to be elucidated. Recent evidences emphasized the major impact of microcirculation on isletβ-cell mass and function. And potentials in islet graft revascularization are crucial for their survival and preserved function in the recipient. In this study, we verified the distinct histological phenotype and functionality of small islets versus large ones both in vitro and in vivo. With efforts to exploring the differences in microcirculation and revascularization of islet grafts, we further evaluated local expressions of angiotensin and vascular endothelial growth factor A (VEGF-A) at different levels. Our findings reveal that, apart from the higher density of insulin-producingβ-cells, small islets express less angiotensin and more angiotrophic VEGF-A. We therefore hypothesized a logical explanation of the small islet superiority for transplantation outcome from the aspects of facilitated microcirculation and revascularization intrinsically in small islets.

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