Michael White

Expression of Mesenchymal and α-Cell Phenotypic Markers in Islet β-Cells in Recently Diagnosed Diabetes

OBJECTIVE Relative contributions of reversible β-cell dysfunction and true decrease in β-cell mass in type 2 diabetes remain unclear. Definitive rodent lineage-tracing studies have identified β-cell dedifferentiation and subsequent reprogramming to α-cell fate as a novel mechanism underlying β-cell failure. The aim was to determine whether phenotypes of β-cell dedifferentiation and plasticity are present in human diabetes. RESEARCH DESIGN AND METHODS Immunofluorescence colocalization studies using classical endocrine and mesenchymal phenotypic markers were undertaken using pancreatic sections and isolated islets from three individuals with diabetes and five nondiabetic control subjects. RESULTS Intraislet cytoplasmic coexpression of insulin and vimentin, insulin and glucagon, and vimentin and glucagon were demonstrated in all cases. These phenotypes were not present in nondiabetic control subjects. CONCLUSIONS Coexpression of mesenchymal and α-cell phenotypic markers in human diabetic islet β-cells has been confirmed, providing circumstantial evidence for β-cell dedifferentiation and possible reprogramming to α-cells in clinical diabetes.

Type 2 Diabetes: The Pathologic Basis of Reversible β-Cell Dysfunction

The reversible nature of early type 2 diabetes has been demonstrated in in vivo human studies. Recent in vivo and in vitro studies of β-cell biology have established that the β-cell loses differentiated characteristics, including glucose-mediated insulin secretion, under metabolic stress. Critically, the β-cell dedifferentiation produced by long-term excess nutrient supply is reversible. Weight loss in humans permits restoration of first-phase insulin secretion associated with the return to normal of the elevated intrapancreatic triglyceride content. However, in type 2 diabetes of duration greater than 10 years, the cellular changes appear to pass a point of no return. This review summarizes the evidence that early type 2 diabetes can be regarded as a reversible β-cell response to chronic positive calorie balance.

Pluripotency-associated stem cell marker expression in proliferative cell cultures derived from adult human pancreas.

The source of new β-cells in adult human pancreas remains incompletely elucidated with recent studies on rodents providing evidence for neogenesis from progenitor cells in addition to self-replication. The aim of this study was to investigate the expression of pluripotency-associated stem cell markers in proliferative cultures derived from adult human pancreas. Human pancreatic tissue was obtained from deceased donors following ethical approval and relative consent. Islet-enriched fraction was separated from the retrieved organ by digestion and density gradient centrifugation. Dissociated cells were seeded in adherent culture forming proliferative islet survivor cells (ISCs). These were characterised at fifth passage by RT-PCR, immunofluorescence staining, FACS, western blot and transfection studies with an OCT4 promoter-driven reporter. Nuclear expression of the pluripotency-associated stem cell marker complex OCT4/SOX2/NANOG was confirmed in ISCs. The phenotype constituted ∼8% of the overall population. OCT4 biosynthesis was confirmed by western blot and activation of an exogenous OCT4 promoter. Co-expression of pluripotency-associated markers has been confirmed in proliferative primary cells derived from adult human pancreas. Further studies are required to elucidate whether these cells possess functional stem cell characteristics and assess potential for differentiation into pancreatic cell lineages including new β-cells.

Loss of end-differentiated β-cell phenotype following pancreatic islet transplantation

Replacement of pancreatic β‐cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life‐threatening hypoglycemia in type 1 diabetes. Although near‐normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β‐cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress‐induced β‐cell dysfunction in type 2 diabetes. Here we report loss of end‐differentiated β‐cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin‐positive cells had lost expression of the end‐differentiation marker, urocortin‐3, or appeared to co‐express the α‐cell marker, glucagon. In contrast, no insulin+/urocortin‐3− cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end‐differentiated phenotype may facilitate β‐cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β‐cell sources, graft sites, and ultimately fully vascularized bioengineered insulin‐secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β‐cell phenotype has been maintained.

Beta cell dedifferentiation: Evidence for ductal progenitors as a source of new beta cells?

Aims: The Diabetes Control and Complications Trial reported that maintained C-peptide production benefits blood glucose control in individuals with Type 1 diabetes. We investigated whether restoration of endogenous C-peptide production by islet transplantation improved blood glucose control, and whether loss of C-peptide below a given threshold reduced clinical benefit. n nMethods: Ten UK islet recipients [age (mean ± SD) 52.3 ± 6.8years, 90% female] had continuous glucose monitoring (CGM) for five to seven days pre-transplant and one, three and six months post-transplant. At each follow-up, graft function was determined by 90min C-peptide during a meal tolerance test (MTT90). Blood glucose 10mmol/l hyperglycaemic. Blood glucose variability was assessed by standard deviation from mean glucose. Outcomes were compared in recipients with MTT90>200pmol/l vs<200pmol/l. n nResults: Islet graft function with MTT90>200pmol/l was established in 80%, 80% and 70% of recipients at one, three and six months post-transplant respectively. Recipients achieving MTT90>200pmol/l spent more time normoglycaemic post-transplant (48.6% pre- vs 80.9%, 77.7%, 82.9% at one, three and six months post-transplant, p 200pmol/l compared with pre-transplant (2.2mmol/l vs 4.1mmol/l, p<0.01) and individuals with MTT90<200pmol/l (4.0mmol/l, p<0.01), who failed to sustain significant improvement in variability compared with pre-transplant (p=0.86). n nConclusions: Stimulated C-peptide>200pmol/l was associated with reduced hyperglycaemia and blood glucose variability post-islet transplant. Therapies aimed at maintaining/restoring endogenous C-peptide production need to consider the thresholds required for clinical benefit.