The KINGS Ins2 +/G32S Mouse: A Novel Model of ß-Cell Endoplasmic Reticulum Stress and Human Diabetes.

Animal models are important tools in diabetes research because ethical and logistical constraints limit access to human tissue. ß-Cell dysfunction is a common contributor to the pathogenesis of most types of diabetes. Spontaneous hyperglycemia was developed in a colony of C57BL/6J mice at King's College London (KCL). Sequencing identified a mutation in the Ins2 gene, causing a glycine-to-serine substitution at position 32 on the B chain of the preproinsulin 2 molecule. Mice with the Ins2 +/G32S mutation were named KCL Ins2 G32S (KINGS) mice. The same mutation in humans (rs80356664) causes dominantly inherited neonatal diabetes. Mice were characterized, and ß-cell function was investigated. Male mice became overtly diabetic at ∼5 weeks of age, whereas female mice had only slightly elevated nonfasting glycemia. Islets showed decreased insulin content and impaired glucose-induced insulin secretion, which was more severe in males. Transmission electron microscopy and studies of gene and protein expression showed ß-cell endoplasmic reticulum (ER) stress in both sexes. Despite this, ß-cell numbers were only slightly reduced in older animals. In conclusion, the KINGS mouse is a novel model of a human form of diabetes that may be useful to study ß-cell responses to ER stress.

Modulation of Rab7a-mediated growth factor receptor trafficking inhibits islet beta cell apoptosis and autophagy under conditions of metabolic stress.

Regenerative medicine approaches to enhancing beta cell growth and survival represent potential treatments for diabetes. It is known that growth factors such as insulin, IGF-1 and HGF support beta cell growth and survival, but in people with type 2 diabetes the destructive effects of metabolic stress predominate and beta cell death or dysfunction occurs. In this study we explore the novel hypothesis that regulation of growth factor receptor trafficking can be used to promote islet beta cell survival. Growth factor signalling is dependent on the presence of cell surface receptors. Endosomal trafficking and subsequent recycling or degradation of these receptors is controlled by the Rab GTPase family of proteins. We show that Rab7a siRNA inhibition enhances IGF-1 and HGF signalling in beta cells and increases expression of the growth factor receptors IGF-1R and c-Met. Furthermore, Rab7a inhibition promotes beta cell growth and islet survival, and protects against activation of apoptosis and autophagy pathways under conditions of metabolic stress. This study therefore demonstrates that Rab7a-mediated trafficking of growth factor receptors controls beta cell survival. Pharmaceutical Rab7a inhibition may provide a means to promote beta cell survival in the context of metabolic stress and prevent the onset of type 2 diabetes.

Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell-Derived Hepatocytes for Cell-Based Therapy.

Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D poly(ethylene glycol)-diacrylate fabricated inverted colloid crystal scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate and remained viable as well as functional upon transplantation into immune competent mice. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications. Stem Cells Translational Medicine 2019;8:124&14.

Impaired Nociception in the Diabetic Ins2+/Akita Mouse.

The mechanisms responsible for painful and insensate diabetic neuropathy are not completely understood. Here, we have investigated sensory neuropathy in the Ins2+/Akita mouse, a hereditary model of diabetes. Akita mice become diabetic soon after weaning, and we show that this is accompanied by an impaired mechanical and thermal nociception and a significant loss of intraepidermal nerve fibers. Electrophysiological investigations of skin-nerve preparations identified a reduced rate of action potential discharge in Ins2+/Akita mechanonociceptors compared with wild-type littermates, whereas the function of low-threshold A-fibers was essentially intact. Studies of isolated sensory neurons demonstrated a markedly reduced heat responsiveness in Ins2+/Akita dorsal root ganglion (DRG) neurons, but a mostly unchanged function of cold-sensitive neurons. Restoration of normal glucose control by islet transplantation produced a rapid recovery of nociception, which occurred before normoglycemia had been achieved. Islet transplantation also restored Ins2+/Akita intraepidermal nerve fiber density to the same level as wild-type mice, indicating that restored insulin production can reverse both sensory and anatomical abnormalities of diabetic neuropathy in mice. The reduced rate of action potential discharge in nociceptive fibers and the impaired heat responsiveness of Ins2+/Akita DRG neurons suggest that ionic sensory transduction and transmission mechanisms are modified by diabetes.

Annexin A1 Is a Key Modulator of Mesenchymal Stromal Cell-Mediated Improvements in Islet Function.

We have previously demonstrated that coculture of islets with mesenchymal stromal cells (MSCs) enhanced islet insulin secretory capacity in vitro, correlating with improved graft function in vivo. To identify factors that contribute to MSC-mediated improvements in islet function, we have used an unbiased quantitative RT-PCR screening approach to identify MSC-derived peptide ligands of G-protein-coupled receptors that are expressed by islets cells. We demonstrated high expression of annexin A1 (ANXA1) mRNA by MSCs and confirmed expression at the protein level in lysates and MSC-conditioned media by Western blot analysis and ELISA. Preculturing islets with exogenous ANXA1 enhanced glucose-stimulated insulin secretion (GSIS), thereby mimicking the beneficial influence of MSC preculture in vitro. Small interfering RNA-mediated knockdown of ANXA1 in MSCs reduced their capacity to potentiate GSIS. MSCs derived from ANXA1(-/-) mice had no functional capacity to enhance GSIS, in contrast to wild-type controls. Preculturing islets with ANXA1 had modest effects on their capacity to regulate blood glucose in streptozotocin-induced diabetic mice, indicating that additional MSC-derived factors are required to fully mimic the beneficial effects of MSC preculture in vivo. These findings demonstrate the feasibility of harnessing the MSC secretome as a defined, noncellular strategy to improve the efficiency of clinical islet transplantation protocols.

Assembly of bioactive multilayered nanocoatings on pancreatic islet cells: incorporation of α1-antitrypsin into the coatings.

A spontaneous multilayer deposition approach for presenting therapeutic proteins onto pancreatic islet surfaces, using a heparin polyaldehyde and glycol chitosan alternating layering scheme, has been developed to enable the nanoscale engineering of a microenvironment for transplanted cells. The nanocoating incorporating α1-antitrypsin, an anti-inflammatory protein, exhibited effective anti-coagulant activities in vitro.