Porcine Insulin Research Articles

Development of an isotope dilution mass spectrometry assay for the quantification of insulin based on signature peptide analysis.

An isotope dilution mass spectrometry (IDMS) method that involves peptide-based protein analysis was developed to accurately quantify insulin. In this study, a signature peptide (GFFYTPK) obtained from tryptic digestion of insulin was selected as a surrogate for insulin. Then, the optimal conditions for signature peptide analysis through mass spectrometry detection and enzymatic digestion were determined. The analytical performance of this method was assessed and validated using porcine insulin-certified reference material. The linear range of the insulin calibration curve ranged from 0.05 ~ 2 mass ratios, with recoveries ranging from 96.15 to approximately 101.15%. The limit of detection was 0.19ng/mL, and the limit of quantification was 0.63ng/mL. The quantitative results corresponded well with a certified value that was obtained from measuring a porcine insulin reference material with amino acid-based IDMS. In addition, the target peptide GFFYTPK can be found in other species of insulin. This method was also applied for the quantification of human insulin-certified reference material. Finally, we applied the method to quantify the concentrations of simulated serum insulin. These findings suggested that this signature peptide-based IDMS approach can accurately quantify insulin levels, can assign a certified value to insulin reference materials, and has the potential to quantify serum insulin with traceable measurements.

Forensic Hypoglycaemia & Neuroglycopenia — A Clinical Legal Social Endocrinology Challenge for 2024, Forensic Law in Hypoglycaemia 4

Forensic Hypoglycaemia & Neuroglycopenia — A Clinical Legal Social Endocrinology Challenge for 2024, Forensic Law in Hypoglycaemia 4

Chromatographic-mass spectrometric analysis of peptidic analytes (2-10kDa) in doping control urine samples.

Peptides with a molecular mass between 2 and 10kDa that are prohibited in elite sports usually require dedicated sample preparation and mass spectrometric detection that commonly cannot be combined with other (lower molecular mass) substances. In most instances, the physicochemical differences are too significant to allow for a generic analytical procedure. A simplification of established and comparably complex analytical approaches is therefore desirable and has been accomplished in the context of this study. With urine samples representing still the most frequently collected doping control specimens, efficient extraction of peptidic analytes from this matrix was a major goal of this method, as demonstrated for the included compounds such as insulins (human, lispro, aspart, glulisine, tresiba, glargine metabolite, bovine insulin, porcine insulin), growth hormone-releasing hormones (sermorelin, CJC-1295, tesamorelin) incl. their respective metabolites, insulin-like-growth factors (long-R3 -IGF-I, R3 -IGF-I, des1-3 -IGF-I), synacthen, gonadorelin and mechano growth factors (human MGF, MGF-Goldspink). Sample preparation and detection are controlled by five internal standards, covering all five included peptide drug categories. Nearly all requirements of the recent technical documents from the World Anti-Doping Agency (WADA) considering their minimum required performance levels (MRPL) are fulfilled, and the method was validated for its utilisation as initial testing procedure in doping controls. Finally, the approach was applied to authentic post-administration study urine samples (for insulins and gonadorelin) in order to provide proof of principle.

#77 : A Micro 3D Printed Device for Intracytoplasmic Sperm Injection Removes the Requirement for a Holding Pipette and Reduces Complexity of the Procedure

Background and Aims: One of the most skilled procedures performed by embryologists is intracytoplasmic sperm injection (ICSI), which takes significant experience to master. Conducting the procedure requires a high level of concentration with training a significant burden on clinical laboratory efficiency. Advancement of a micro 3D printed device to hold oocytes has removed the requirement for a holding pipette, reducing the complexity of the ICSI procedure. This study shows that the microICSIÔ device reduces the technical difficulty of ICSI, improving the speed and precision of the procedure. Method: ICSI was performed conventionally (C-ICSI) and within the microICSIÔ device on abattoir derived porcine denuded oocytes matured in vitro for 39-44 h in NCSU-23 medium containing eCG, hCG, porcine follicular fluid, EGF and insulin. Timing data was captured by video recording for pipette setup and injection time per oocyte for two embryologists and compared between each technique. Oocyte injection time was taken from when a sperm loaded injection pipette was in focus with the oocyte until the injection pipette was removed from the oocyte. Results: Results were compared between C-ICSI and microICSIÔ (Mean ± SD). Pipette setup time was reduced from 278 ± 21 seconds to 109 ± 3 seconds by removing the holding pipette. During the procedure, absence of a holding pipette resulted in embryologists adjusting their hands on average only 3 times during oocyte injection compared to 10 times for C-ICSI when moving between microinjector, pipette and microscope controllers. Oocyte traceability was also improved due to the microICSIÔ device’s numbered array. Embryologist’s injection time was reduced from 42 ± 12 seconds for C-ICSI to 14 ± 5 seconds for microICSIÔ per oocyte. Conclusion: The microICSIÔ device reduces pipette set-up time and the complexity of the ICSI procedure, reducing the time required for oocyte injection by half.

Characterisation of transgenic pigs expressing a human T cell-depleting anti-CD2 monoclonal antibody.

Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell-mediated rejection in baboons. Local expression of a depleting anti-CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock-in transgenic pigs expressing the chimeric anti-CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock-in pigs in which MHCIP was replaced by the β-cell-specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. A PIP-diliximab knock-in construct was prepared and validated by transfection of NIT-1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock-in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP-diliximab and PIP-diliximab knock-in pigs was characterised at the mRNA and protein levels using RT-qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP-diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice. NIT-1 cells stably transfected with the PIP-diliximab knock-in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in β cells. PIP-diliximab knock-in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP-diliximab pigs, but was not detectable in PIP-diliximab pigs. Likewise, diliximab was present in the serum of MHCIP-diliximab pigs, at a mean concentration of 1.8 μg/mL, but was not detected in PIP-diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP-diliximab pigs but not of PIP-diliximab pigs. Whole genome sequencing (WGS) of a PIP-diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock-in clone used to generate the PIP-diliximab pigs. Islet xenografts from neonatal MHCIP-diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. Diliximab was widely expressed in MHCIP-diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP-diliximab pigs is sufficient to prevent T cell-mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP-diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock-in cells used for SCNT.

Desupersaturation dynamics in solutions with applications to bovine and porcine insulin crystallization

Evolution of crystal ensembles in supersaturated solutions is studied at the initial and intermediate stages of bulk crystallization. An integro-differential model includes fluctuations in crystal growth rates, initial crystal-size distribution and arbitrary nucleation and growth kinetics of crystals. Two methods based on variables separation and saddle-point technique for constructing a complete analytical solution to this model are considered. Exact parametric solutions based on these methods are derived. Desupersaturation dynamics is in good agreement with the experimental data for bovine and porcine insulin. The method based on variables separation has a strong physical limitation on exponentially decaying initial distribution and leads to the distribution function increasing with time. The method based on saddle-point technique leads to a dome-shaped crystal-size distribution function decreasing with time and has no strong physical limitations. The latter circumstance makes this method more reasonable for describing the kinetics of bulk crystallization in solutions and melts.

Development of a Web App to Convert Blood Insulin Concentrations among Various Immunoassays Used in Horses.

The measurement of the blood insulin concentration, and comparison to cut-offs, is essential in diagnosing insulin dysregulation, a common equine endocrinopathy. However, different insulin assays provide disparate results. We aimed to ease comparison between assays by compiling original and published data into a web app to convert insulin measurements from one assay to another. Data were available for ADVIA Centaur insulin chemiluminescent immunoassay (CLIA), Beckman Coulter insulin radioimmunoassay (RIA), Immulite 1000 CLIA, Immulite 2000 CLIA, Immulite 2000 XPi CLIA, Mercodia equine insulin enzyme-linked immunosorbent assay (ELISA), and Millipore porcine insulin RIA. Linear models were fitted for 13 assay pairs using non-decreasing splines, and integrated into this app. Assay comparisons including data from several studies showed a lower performance. This indicates technical variation between laboratories, which has not been described before, but is relevant when diagnostic measurements and cut-offs are provided by different laboratories. Nevertheless, the models' overall high performance (median r2 = 0.94; range 0.57-1.00) supports their use to interpret results from diagnostic insulin measurements when the reference assay is unavailable, and to compare values obtained from different assays.

P-259 Intracytoplasmic sperm injection conducted within a micro 3D printed device that requires no holding pipette or vacuum reduces porcine oocyte invagination during microinjection

Abstract Study question Can the use of a custom-designed and 3D printed microICSI™ device reduce the degree of oocyte invagination during intracytoplasmic sperm injection (ICSI)? Summary answer Invagination was significantly less in porcine oocytes when ICSI was performed in microICSI™ device compared to conventional ICSI. What is known already ICSI is a difficult procedure for embryologists to master, yet until recently, technically little had changed. In particular, the holding pipette (HP) with vacuum to hold the oocyte has not been surpassed. However, a source of shear-stress during ICSI of oocytes is the invagination of the zona pellucida and cytoplasm that occurs during microinjection, due to the pressure of the injection pipette against the small surface area of the HP. A recent study demonstrated microinjection within a two-piece 3D printed device. Here we evaluated a design refinement that displaced injection pressure more evenly across the oocyte to improve embryo quality. Study design, size, duration A control (conventional ICSI) and study group (microICSI™) were compared over three replicates investigating the level of invagination (3-point score, 1 is lowest and 3 is greatest) during ICSI. Incidence of cytoplasmic lysis was also recorded 24 h following ICSI. Participants/materials, setting, methods Porcine oocytes collected by follicle aspiration of abattoir-sourced ovaries were matured for 39-44 h in NCSU-17 medium supplemented with eCG, hCG, porcine follicular fluid, EGF and insulin. Mature oocytes, stripped of cumulus, were randomly allocated to either conventional ICSI or microICSI™. ICSI was performed, with treatment order randomised, and the injected oocytes were cultured in NCSU-17 culture medium at 39 C. Main results and the role of chance The microICSI™ device was modelled as a one-piece structure with contoured surfaces within a microwell array to support the oocyte during injection without the requirement for a holding pipette. Two-photon polymerisation 3D micro printing was used to print and assess multiple prototype geometries to optimise the support of the oocyte during ICSI. Over the three replicates (n = 10-20/treatment/replicate), the degree of invagination was consistently lower in the microICSI™ group compared with conventional ICSI (Mean ± SEM): Replicate 1: 1.40 ± 0.16 vs. 2.00 ± 0.19; Replicate 2: 1.20 ± 0.11 vs. 2.00 ± 0.13; Replicate 3: 1.42 ± 0.12 vs. 1.78 ± 0.15, with a paired t-test showing a significant difference between the two groups (p = 0.035). The incidence of lysis was not significantly different between the two treatments (ranging from 15-20% across both groups and varied between replicates). Limitations, reasons for caution The use of a porcine ICSI model may not sufficiently replicate what occurs with human ICSI as human oocytes experience greater levels of invagination (typically scored out of 4 rather than 3). Wider implications of the findings Reducing the invagination due to the injection process may reflect a less stressful procedure, with improvement to fertilization rates and embryo quality. Trial registration number not applicable

Regulatory Verification by Health Canada of Content in Recombinant Human Insulin, Human Insulin Analog, and Porcine Insulin Drug Products in the Canadian Market Using Validated Pharmacopoeial Methods Over Nonvalidated Approaches.

For diabetes mellitus treatment plans, the consistency and quality of insulin drug products are crucial for patient well-being. Because biologic drugs, such as insulin, are complex heterogeneous products, the methods for drug product evaluation should be carefully validated for use. As such, these criteria are rigorously evaluated and monitored by national authorities. Consequently, reports that describe significantly lower insulin content than their label claims are a concern. This issue was raised by a past publication analyzing insulin drug products available in Canada, and, as a result, consumers and major patient organizations have requested clarification. To address these concerns, this study independently analyzed insulin drug products purchased from local Canadian pharmacies-including human insulin, insulin analogs, and porcine insulin-by compendial and noncompendial reversed-phase high-performance liquid chromatography (RP-HPLC) methods. We demonstrated the importance of using methods fit for purpose when assessing insulin quality. In a preliminary screen, the expected insulin peak was seen in all products except two insulin analogs-insulin detemir and insulin degludec. Further investigation showed that this was not caused by low insulin content but insufficient solvent conditions, which demonstrated the necessity for methods to be adequately validated for product-specific use. When drug products were appropriately assessed for content using the validated type-specific compendial RP-HPLC methods for insulin quantitation, values agreed with the label claim content. Because insulin drug products are used daily by over a million Canadians, it is important that researchers and journals present data using methods fit for purpose and that readers evaluate such reports critically.

In vitro Study of Antioxidant, Antidiabetic and Antimicrobial Activities of <i>Solanum Torvum</i> Root

Diabetes mellitus is a metabolic disorder with characteristics of multiple aetiology of chronic hyperglycaemia. These effects include dysfunction and long-term degeneration of various organs. Characteristics of diabetes mellitus include polyuria, blurred vision, thirst, and weight loss. Many types of insulin are available, for example porcine insulin, beef insulin, and human insulin. Weight gain and hypoglycaemia are the most adverse effects of using insulin. Insulin binds to sulfonylurea receptors on β-cell plasma membranes. In this study, ethanolic extract of Solanum torvum root was used to study ATP-sensitive potassium channels, which lead to depolarized cell membranes. The ethanolic extract was also analysed for antioxidant activities by free radical 1,1-diphenyl-2-picryl hydrazyl (DPPH) scavenging method. The different concentration of extracts exhibited varying result of radical scavenging activity using DPPH as substrate. The root of Solanum torvum exhibited significant α-amylase (79.56%) and α-glucosidase (80.58%) inhibitory activities at the concentration 100 μg/ml respectively when compared with standard acarbose drug. It also showed a good antioxidant activity with 65.47% when compared with standard ascorbic acid. The ethanolic extract of Solanum torvum root has a potential antimicrobial activity against Staphylococcus aureus and Candida tropicalis. Thus, Solanum torvum is a better source for natural antioxidant, anti-diabetic and anti-microbial activities due to presence of natural compounds in it.

Nutrient Uptake by the Hindlimb of Growing Pigs Treated with Porcine Somatotropin and Insulin

The shift in nutrient partitioning induced by porcine somatotropin (pST) is accompanied by a decrease in insulin sensitivity for whole-body glucose uptake. The relative contribution of metabolic changes in the hindlimb was investigated in eight pigs (55 kg) that had received recombinant pST (120 μg/kg) or excipient (control) for 7 d. Uptake of metabolites by the hindlimb was measured under basal conditions and during hyperinsulinemic-euglycemic clamps at low [14 ng/(kg·min)] and high [360 ng/(kg·min)] insulin infusion rates. Dextrose infusion rate required to maintain euglycemia was used as an index of whole-body glucose uptake in response to exogenous insulin. Effects of pST on hindlimb and whole-body glucose uptake were evident only at physiological levels of insulin (basal and low insulin infusion rate). During the low rate of insulin infusion, dextrose infusion rate was 79% lower for pST-treated pigs and glucose uptake by the hindlimb was 59% lower compared with control pigs. The decrease in glucose uptake by the hindlimb was entirely accounted for by the estimated reduction in glucose utilization by adipose tissue of the hindlimb. Glucose:oxygen quotients were reduced during basal (57%) and low insulin infusion (63%) with pST treatment, indicating a change in the pattern of substrate utilization. This is consistent with the concept that pST directs nutrients away from adipose and towards muscle growth by altering the response of tissues to homeostatic signals such as insulin.

Pigs with an INS point mutation derived from zygotes electroporated with CRISPR/Cas9 and ssODN.

Just one amino acid at the carboxy-terminus of the B chain distinguishes human insulin from porcine insulin. By introducing a precise point mutation into the porcine insulin (INS) gene, we were able to generate genetically modified pigs that secreted human insulin; these pigs may be suitable donors for islet xenotransplantation. The electroporation of the CRISPR/Cas9 gene-editing system into zygotes is frequently used to establish genetically modified rodents, as it requires less time and no micromanipulation. However, electroporation has not been used to generate point-mutated pigs yet. In the present study, we introduced a point mutation into porcine zygotes via electroporation using the CRISPR/Cas9 system to generate INS point-mutated pigs as suitable islet donors. We first optimized the efficiency of introducing point mutations by evaluating the effect of Scr7 and the homology arm length of ssODN on improving homology-directed repair-mediated gene modification. Subsequently, we prepared electroporated zygotes under optimized conditions and transferred them to recipient gilts. Two recipients became pregnant and delivered five piglets. Three of the five piglets carried only the biallelic frame-shift mutation in the INS gene, whereas the other two successfully carried the desired point mutation. One of the two pigs mated with a WT boar, and this desired point mutation was successfully inherited in the next F1 generation. In conclusion, we successfully established genetically engineered pigs with the desired point mutation via electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.

Human growth hormone and insulin, from tissue extracts to recombinant DNA technology, their biosimilars and analogues in treatment of Endocrine Disorders

Forty years ago, recombinant DNA technology caused a revolution in the way growth disorders and diabetes could be treated. Bacteria or human cells were modified so as to be able to produce human hormones instead of relying on cadaveric material in the case of growth disorders and porcine insulin in the case of diabetics. The new technology also removed the risk of contracted hepatitis or fatal Creutzfeldt Jacob disease. A signal peptide was used to transport the new protein into the periplasmic space giving rise to an exact, correctly folded version of monomeric human growth hormone. More eligible patients could be treated, resulting not only in a large number of clinical trials. Daily tiny subcutaneous injections were found to be much more effective than twice or thrice weekly in the treatment of children and adults, The progress for recombinant human insulin turned out to be somewhat different. Insulin is a more complicated protein, with an Alpha and Beta chain joined by two disulphide bridges, but is produced in the human pancreas as a single chain, proinsulin, the C peptide is removed to make the active molecule. Recombinant insulin development was in a way the opposite to growth hormone since human insulin acts faster than porcine. Short fast and long-acting analogues have been developed all to improve the treatment regimens for both type 1 and type 2 diabetics, A similar development is now taking place in the growth hormone field. Depot preparations for weekly administration are under development to achieve better compliance. One recently approved depot uses an analogue, modified in one amino acid of the peptide chain to permit acylation and albumin binding. In a way this is a retrograde step and it remains to be seen if the patient’s growth and metabolic responses are comparable to the daily administration of tiny amount of recombinant natural sequence hormone over several years of treatment.

Owner preference for insulin delivery device and glycaemic control in diabetic dogs.

To assess treatment satisfaction and owner preference for two delivery devices (VetPen, MSD Animal Health, and U40 insulin syringes) and the effect on glycaemic control in diabetic dogs treated with porcine insulin zinc suspension. Randomised prospective cross-over study with two arms, each of 8 weeks. Twenty client-owned diabetic dogs on insulin treatment by U40 syringe were enrolled. Dogs were randomly assigned to receive insulin by syringe or pen injector for 2 months, followed by 2 months of the other injection method. Treatment satisfaction and owners' insulin delivery device preference were assessed using a questionnaire. Glycaemic control was assessed using a clinical score, serum fructosamine and glycated haemoglobin (HbA1c%) at the time of the enrolment (T0) and the end of each arm of treatment (T2 and T4). Treatment satisfaction differed for the two types of the delivery device when the order that each device was used was taken into consideration. Owners who used the syringe first did not have a significant preference for an injection device. In contrast, owners who used the pen injector first expressed a significant preference for VetPen compared to syringes. No significant differences in the number of dogs of Groups 1 and 2 with good and poor glycaemic control at T2 and T4 were detected. Overall treatment satisfaction and preference for the two delivery methods were similar. However, VetPen was preferred by owners who were randomised to use this device first. Glycaemic control did not appear to be affected by the insulin delivery device used.

P12.06: Effect of Supplementing Taurine to the Developed Lowtemperature Islet Preservation Solution

Introduction: Pancreatic islet transplantation has recently emerged as one of the most promising therapeutic approaches for improving glycemic control in type 1 diabetes patients. However, one of the problems with islet transplantation is that it is impossible to culture the isolated islet for extended periods to while recipients are selected, tested and prepared for surgery. To address this problem, we developed the islet preservation solution, and to improve the function of the preservation solution, we added taurine and conducted a preservation experiment. Taurine is antioxidant activity and it is not a classical free radical scavenger. Therefore, its mechanism remains unclear but it controls osmotic pressure. Our hypothesis is that taurine supplementation can improve islet recovery and enhance islet function after low temperature preservation. Methods: CMRL 1066 medium (Corning) was used as a control group, and a comparison group was prepared some compounds OPTI(Optipharm presevation) solution (with taurine) and OPTI-T solution (without taurine). To confirm the preservation effect of these solutions, we isolated islet from adult Yucatan pig’s pancreas using standard enzymatic digestion (Nordmark) followed by Ficoll purification, these islets were preserved in a humidified tissue culture incubator with preservation solutions and preserved in 4℃ for up to 4 weeks. We compared islet survival in groups of islets. To islet viability and islet functionality, we utilized Glucose Stimulated Insulin Secretion (GSIS) for measuring insulin release, calculated stimulation index (SI) and AO/PI viability staining for during the preservation period. Results: Highest islet recovery was preserved in the group of pig islets cultured with Taurine as compared to the group of islets hypothermically preserved in standard tissue culture media over the 4 week preservation period (p<0.001 using ANOVA). The porcine islet viability/cell number ratio in the OPTI solution maintained a high survival rate for up to 2 weeks (94%). In contrast, porcine islet in OPTI-T solution and CMRL 1066 supplemented media significantly reduced viability after 1 weeks. OPTI solution (with taurine) was significantly higher than OPTI-T solution (without taurine) and CMRL 1066 during 4 weeks after preservation. Glucose stimulation insulin secretion test was performed for functional analysis. As a result, a level of porcine insulin was higher during 2 weeks in the OPTI solution than OPTI-T solution and CMRL 1066. But after 3 weeks preservation, insulin level in high glucose, significantly decreased. Therefore, the viability and functionality of islets were improved by adding taurine to the developed preservation solution. Conclusion: Taurine supplementation has been shown to highest islet recovery, viability and insulin secretion level for 2 weeks. Based on this data, we have performed 2 weeks cold storage of isolated islets for current clinical islet transplantation. Ministry of trade industry and energy, KEIT 20011276, NTIS 1415172664.

Probing for peptidic drugs (2-10kDa) in doping control blood samples.

Bioactive peptides with a molecular mass between 2 and 10kDa represent an important class of substances banned in elite sports, which has been recognized with an increasing number and variety of substances by anti-doping organizations. Also, the annually renewed list of prohibited substances of the World Anti-Doping Agency (WADA) explicitly mentions more and more of these peptides, and efficient testing procedures are required. Even under simplified sample preparation conditions, liquid chromatography coupled to high-resolution mass spectrometry (with resolution properties>100,000 full width at half maximum) offers suitable conditions for this task and can therefore be used as an initial testing procedure. In contrast to urine, blood analysis essentially relies on the detection of intact peptide hormones, and the expected concentrations are commonly higher in blood samples than in urine. This facilitates the analysis, and a generic sample preparation by means of mixed-mode solid-phase extraction could be realized in this study. Co-extraction and analysis of several different peptides such as insulins (human, lispro, aspart, glulisine, tresiba, detemir, glargine, bovine insulin and porcine insulin), growth hormone releasing hormones (sermorelin, CJC-1295 and tesamorelin), insulin-like growth factors (long-R3-IGF-I, R3-IGF-I and Des1-3-IGF-I) and mechano growth factors (human MGF and MGF-Goldspink) with criteria that fulfil the requirements of the WADA documents (TD2022 MRPL) for doping controls. The proof of principle was shown by the analysis of post administration samples after treatment with synthetic insulin analogues.

Bioabsorption of Subcutaneous Nanofibrous Scaffolds Influences the Engraftment and Function of Neonatal Porcine Islets.

The subcutaneous space is currently being pursued as an alternative transplant site for ß-cell replacement therapies due to its retrievability, minimally invasive procedure and potential for graft imaging. However, implantation of ß-cells into an unmodified subcutaneous niche fails to reverse diabetes due to a lack of adequate blood supply. Herein, poly (ε-caprolactone) (PCL) and poly (lactic-co-glycolic acid) (PLGA) polymers were used to make scaffolds and were functionalized with peptides (RGD (Arginine-glycine-aspartate), VEGF (Vascular endothelial growth factor), laminin) or gelatin to augment engraftment. PCL, PCL + RGD + VEGF (PCL + R + V), PCL + RGD + Laminin (PCL + R + L), PLGA and PLGA + Gelatin (PLGA + G) scaffolds were implanted into the subcutaneous space of immunodeficient Rag mice. After four weeks, neonatal porcine islets (NPIs) were transplanted within the lumen of the scaffolds or under the kidney capsule (KC). Graft function was evaluated by blood glucose, serum porcine insulin, glucose tolerance tests, graft cellular insulin content and histologically. PLGA and PLGA + G scaffold recipients achieved significantly superior euglycemia rates (86% and 100%, respectively) compared to PCL scaffold recipients (0% euglycemic) (* p < 0.05, ** p < 0.01, respectively). PLGA scaffolds exhibited superior glucose tolerance (* p < 0.05) and serum porcine insulin secretion (* p < 0.05) compared to PCL scaffolds. Functionalized PLGA + G scaffold recipients exhibited higher total cellular insulin contents compared to PLGA-only recipients (* p < 0.05). This study demonstrates that the bioabsorption of PLGA-based fibrous scaffolds is a key factor that facilitates the function of NPIs transplanted subcutaneously in diabetic mice.

307.4: Subcutaneous Bioabsorption of Nanofibrous Scaffolds Influence the Engraftment and Function of Neonatal Porcine Islets Xenografts in Mice

Dr. Korbutt and Dr. Pepper Introduction: The subcutaneous space is currently being pursued as an alternative transplant site for human pancreatic islets, stem cell and xenogeneic derived-islets, due to its retrievability, minimally invasiveness, accommodation of large transplant volumes, and potential for monitoring graft function. However, transplantation of islets into an unmodified subcutaneous niche fails to reverse diabetes due to a lack of adequate blood supply. Biomaterial nanofibrous scaffolds can be functionalized to enhance the host tissues integration, provide a platform for the delivery of pro-engraftment growth factors, and immunomodulatory agents. Method: Herein, we utilize poly (ε-caprolactone) (PCL) and poly (lactic-co-glycolic acid) (PLGA) polymers to make nanofibrous scaffolds and functionalized with bioactive peptides, to prime the subcutaneous space into a more suitable environment. We implanted the nylon catheter (2 cm length, 6 French diameter) (deviceless space, DL), and nanofibrous scaffolds (scaffold was wrapped around the nylon catheter) such as PCL, PCL+RGD+VEGF (PCL+R+V), PCL+RGD+Laminin (PCL+R+L), PLGA and PLGA+Gelatin (PLGA+G) into the subcutaneous space of immunodeficient B6.129S7-Rag1tm1Mom/J mice for four weeks to create a prevascularized space. After 4 weeks, DL and scaffolds implanted mice and those intended for kidney capsule (KC) implantation were rendered diabetic by intraperitoneal injection of 180 mg/kg streptozotocin. Subsequently, neonatal porcine islets (3000 NPI) were transplanted under the KC or within the subcutaneous DL and scaffolds. Graft function was evaluated by monitoring non-fasting blood glucose, stimulated porcine insulin measurement, intraperitoneal glucose tolerance test, and histochemical analysis. Results: Our preliminary scaffolds implantation (no cells) study demonstrates that PCL, PCL+R+V and PCL+R+L scaffolds did not absorb and partially integrated with the host tissues (PCL based scaffolds remain intact at the implanted site) whereas PLGA and PLGA+G scaffolds were completely absorbed and integrated with the host tissues (no remnants of PLGA based scaffolds at the implanted site), and we also observed that there were numerous blood vessels innervated in and around these scaffolds. Compared with peptide functionalized PCL scaffolds, PLGA and PLGA+G fibrous scaffolds with NPI resulted in 86% and 100% euglycemia (*p< 0.05, **p<0.01 respectively), superior glucose clearance (*p<0.05) and greater stimulated porcine insulin secretion (*p<0.05). Moreover, PLGA and PLGA+G scaffolds exhibited comparable graft functions with the positive controls (DL and KC)(p>0.05). Conclusion: Our study demonstrates that PLGA based fibrous scaffolds facilitates the engraftment and function of NPIs in the subcutaneous space of diabetic mice. These collective data emphasize the support of biomaterial implants on cellular graft function in the subcutaneous space for clinical islet xenotransplant applications in a near future.

A century of insulin: from lifesaving discovery to a journey of scientific evolution!

AbstractBefore 1922, treatments for type 1 diabetes were limited, including the Allen plan (a diet based on minimising calorific intake), and the prognosis was bleak. Although the pancreas was already thought to produce a substance that, when lacking, resulted in diabetes it was not until the unlikely combination of an orthopaedic surgeon, Frederick Banting, and a medical student, Charles Best, came together with the guidance and support of physiologist Prof John Macleod that progress into clinical practice became a reality. Initial laboratory work in dogs confirmed the biological effect of the endocrine function of the pancreas. The realisation that insulin could be extracted from bovine pancreas made it a realistic prospect for treatment, but only after the involvement of James Collip, a biochemist who helped refine the purification process. The first injection of insulin as a treatment happened on 11 January 1922. Since then, it has been a scientific journey driven by the need for large‐scale production, processes allowing initially for better extraction and purification of bovine, then porcine insulin, before recombinant DNA technology allowed for the production of human insulin from 1978. Throughout the use of animal and human insulins, research efforts have concentrated on finding ways of prolonging the insulin time‐action profile to meet basal insulin requirements while at the same time shortening it to be more convenient for prandial insulin needs. The development of insulin analogues from 1996 has allowed an even better ability to mimic normal physiological function with longer‐acting basal insulins and shorter‐acting prandial insulins. This approach will continue to be a driver for evolution in addition to exploring the possibility of different routes of delivery. Copyright © 2021 John Wiley &amp; Sons.

The in vivo Tetrahymena thermophila extracellular glucose drop assay for characterization of mammalian insulin activity

Insulin activity is generally determined by an in vivo rabbit blood glucose drop assay in research and industriel laboratories. The humane experimental techniques imply the use of alternative invertebrate organisms in place of animals, known as replacement rule of the 3Rs. In this study, we report an alternative in vivo extracellular glucose drop assay using unicellular invertebrate Tetrahymena thermophila to replace the use of rabbit and mouse. This assay has four major steps; growing cells, starving cells, treatment of cells and measurement of glucose drop. In this assay, 0.2 mg/ml of human, porcine and bovine insulins dropped extracellular glucose level to 16%, 14% and 12%, respectively in ten minutes. In addition, mammalian insulins respectively increased the cell area about 19%, 15%, and 16% at 6th hour with statistically significant effect on the cell growth, but not in the cell viability. The results showed that the in vivo Tetrahymena thermophila extracellular glucose drop assay could be used as an alternative assay to replace the mouse or the rabbit insulin blood glucose drop assay.