Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet β cells. However, the role and sequence of β cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of β cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, β cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained β cell volume further declines. These results indicate that dysfunction of persisting β cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Diabetes mellitus is amongst the leading causes of morbidity and mortality in the world, affecting young, adult and old people. Beta cell replacement therapy for insulin delivery remains the ultimate remedy for diabetes. However, insufficient donor pancreas and the use of immunosuppressive drugs prevent the wide-spread of this therapy. Other avenues of self generated beta cells within the organ itself need to be explored. Therefore, understanding the chronobiology of cellular mechanisms in the lineage of beta cell induced neogenesis is a valuable tool in improving beta cell replacement in patients with diabetes. The aim of this study was to induce recapitulation of the morpho-genetic sequence of endocrine cells development in the pancreas of rats after the pancreatic duct ligation (PDL) procedure. Serial sections of PDL tissues of the pancreas were obtained from 78 Sprague- Dawley rats and were assessed morphologically. The immunofluorescent tissues were statistically analysed using a computerized morphometry technique. The protein expression indices of Caspase3, Insulin, Pdx1, Ngn3, NeuroD and Pax6 were quantified. The efficiency levels of coexpression of these homeodomain proteins separately with insulin were defined by the ratio of the mean value of insulin expression to the mean value of their respective protein expression. The morphological changes were characterized by the appearance of granulated acinar cells at 6 hours post-PDL and the proliferation of endocrine tissues from 84 hours through to 120 hours. The morpho-immunofluorescent evaluation showed the highest immunoreactivity of Caspase3 and Pdx1 at 6 hours, Ngn3 at 36 hours, Pax6 and insulin at 84 hours while NeuroD expression was at 120 hours. The immunohistofluorescent analysis showed that caspase3 and Pdx1 were the first to be expressed at 6 hours while the insulin and NeuroD expression appeared later at 84 hours and 120 hours, respectively. However, Pax6 expression was continuous across time periods post-PDL, while Ngn3 expression showed a peak at 36 hours. The efficiency (highest and earliest expression) of co-expression of all these homeodomain proteins with insulin was restricted between 12 hours and 24 hours. The optimal efficiency was at 12 hours by Ngn3 with insulin. A good efficiency was shown for Pdx1 with insulin, NeuroD with insulin and Pax6 with insulin at 12 hours and 24 hours, respectively. A low efficiency was observed for insulin and caspase3 co-expression at 24 hours. This study suggests that for transplantation, PDL tissues harvested at an early time post-PDL (between 12 and 24 hours) could yield a higher success rate; the study also provides evidence for a connection between morphological changes in the PDL pancreas and the protein synthesis necessary for the lineage of endocrine cell development. / AFRIKAANSE OPSOMMING: Diabetes Mellitus resorteer onder die vernaamste oorsake van morbiditeit en mortaliteit wêreldwyd, en tuister jongmense, volwassenes en bejaardes. Daar bestaan egter ‘n wêreldwye tekort aan skenkerorgane met immuun-onderdrukingsterapie as ondersteuningsbehandeling. Beta-sel vervangingsterapie, vir die voorsiening van insulien, bly daarom die voorkeur behandeling vir die siekte wat noodsaak dat die wetenskap kyk na alternatiewe behandelingsregimens wat meganismes rondom orgaanregenerasie insluit. Begrip van die chronobiologie van die sellulêre meganismes betrokke rondom beta-sel ontwikkeling mag waardevolle lig werp op die neogenese van beta-selle wat gevolglik daartoe mag lei dat beta-sel vervanging as ‘n moontlike behandelingsterapie oorweeg mag word vir pasiënte met suikersiekte. Die oogmerk van hierdie studie is om die rekapitulasie van die morfo-genetiese volgorde van die endokriene pankreas na afbinding van die pankreasbuis te bepaal. Pankreasbuis afbinding is op 78 Sprague-Dawley laboratorium rotte onder algemene narkose uitgevoer, die pankreas is na voorafbepaalde tydsvakke verwyder en in histologiese seriesnitte gesny. Snitte is immunositochemiese gekleur en morfometries assesseer. Die afskeidingsindeks vir selboodskappers vir Caspase3, Insulien, Pdx1, Ngn3, NeuroD en Pax6 is kwantifiseer. Die gelyktydige afskeiding van elk van bogenoemde boodskappers tesame met insulien is omskryf as ‘n verhouding tot mekaar en in terme van dié van insulien. Die morfologiese verandering in die weefsel bespeur is gekenmerk deur die verskyn van gegranuleerde asinêre selle ses (6) ure na buisafbinding en die proliferasie van endokriene weefsel vanaf vier-en-tagtig (84) ure deurlopend tot een-honderd-en-twintig (120) ure. Die morfo-immunofluoresserende evaluering toon dat Caspase3 en Pdx1 by 6 uur die hoogste is, die van Ngn3 by 36 ure, Pax6 en insulien by 84 ure en NeuroD by 120 ure. Verder toon die analise dat Caspase3 en Pdx1 rondom 6 ure hul verskyning gemaak het terwyl dié van insulien en NeuroD eers rondom 84 tot 120 uur verskyn het. Die verskyning van Pax6 het deurlopend regoor al die tydsduurtes verskyn en Ngn3 het rondom 36 uur sy hoogste vlak bereik. Die gelyktydige uitdrukking van homeodomein proteïene tesame met insulien het slegs tussen die tydperke van 12 en 24 ure plaasgevind. Die uitdrukking van Pdx1 met insulien, NeuroD met insulien en Pax6 met insulien het almal tussen 12 en 24 ure plaasgevind. Caspase3 tesame met insulien is slegs by die 24 uur tydsperiode bespeur. Vir die oorplant van pankreas weefsel wat aan buisafbinding onderwerp is suggereer hierdie studie dat die geskikste tyd vir die oes van endokriene weefsel liewer vroeër (12 to 24 ure) as later uitgevoer behoort te word. Verder wil dit voorkom of hierdie tydsperiode ook die hoogste seltelling lewer. Die studie lewer waardevolle inligting oor die verwantskap tussen die morfologiese veranderings wat na buisafbinding plaasvind en die proteïen sintese wat sel-opvolgontwikkeling bevorder.