LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic ß cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.

Donor age significantly influences the Raman spectroscopic biomolecular fingerprint of human pancreatic extracellular matrix proteins following collagenase-based digestion.

Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI). However, age-related differences in pancreatic matrix digestion have not been studied in detail at the molecular level. To address this, we investigated ECM digestion in purified ECM proteins and in pancreatic tissue sections from younger (≤30 years; n = 5) and older (>55 years; n = 5) BMI matched donors, using Raman microspectroscopy (RMS). The Raman spectral profiles for purified collagens I, IV, VI and laminins were significantly altered following controlled enzyme treatment. Pancreatic cryosections were treated with Serva collagenase, NP, or the two enzymes combined, at clinically relevant concentrations. RMS demonstrated that the ECM at the islet-exocrine interface was differentially digested with respect to donor age. The action of collagenase was affected to a greater extent than NP. RMS is a powerful, marker-independent technology for characterising the human pancreatic ECM and demonstrating differences between donor types. Ongoing detailed studies using RMS will assist the development of donor-specific enzyme blends, increasing the overall success of human islet isolation and benefiting many people with type 1 diabetes worldwide. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally invasive treatment, which can reverse Type 1 Diabetes Mellitus (T1DM) in selected patients. Islets of Langerhans are extracted (isolated) from the exocrine tissue of human donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current enzymes are ineffective at digesting pancreases from younger donors. Using Raman microspectroscopy we demonstrate that donor age affects the enzymatic digestion of the pancreatic ECM at the molecular level. Collagenase activity is affected to a greater extent than NP. These findings will assist the development of donor-specific enzymes, thereby increasing the overall success of islet isolation and benefiting many people with T1DM worldwide.

Development of a Simple In Vitro Assay to Assess Digestion of the Extracellular Matrix of the Human Pancreas by Collagenase Enzyme Blends.

Despite huge advances in the field of islet transplantation over the last two decades, current islet isolation methods remain suboptimal, with transplantable yields obtained in less than half of all pancreases processed worldwide. Successful islet isolation is dependent on the ability of collagenase-based enzyme blends to digest extracellular matrix components at the islet-exocrine interface. The limited availability of donor pancreases hinders the use of full-scale islet isolations to characterize pancreas digestion by different enzyme components or blends, or allow the influence of inter-pancreatic variability between donors to be explored. We have developed a method that allows multiple enzyme components to be tested on any one pancreas. Biopsies of 0.5 cm3 were taken from seven standard (age ≥45) and eight young (age ≤35) pancreases. Serial cryosections were treated with Serva collagenase, neutral protease (NP), or the two enzymes together at clinically relevant concentrations. Following digestion, insulin and either collagen IV or laminin-α5 were detected by immunofluorescent labeling. Protein loss at the islet-exocrine interface was semi-quantified morphometrically, with reference to a control section. Differential digestion of the two proteins based on the enzyme components used was seen, with protein digestion significantly influenced by donor age. Treatment with collagenase and NP alone was significantly more effective at digesting collagen IV in the standard donor group, as was the NP mediated digestion of laminin-α5. Collagenase alone was not capable of significantly digesting laminin-α5 in either donor group. Combining the two enzymes ameliorated the age-related differences in the digestion of both proteins. No significant differences in protein loss were detected by the method when analyzed by two independent operators, demonstrating the reproducibility of the assay. The development of this simple yet reproducible assay has implications for both enzyme batch testing and identifying inter-donor digestion variability, while utilizing small amounts of both enzyme and human tissue.