New Insight into Metformin Mechanism of Action and Clinical Application

Metformin is the first-line medication for Type 2 diabetes (T2D) treatment, and it is the only US FDA approved oral antidiabetic medication for pediatric patients with T2D 10 years and older. Metformin is also used to treat polycystic ovary syndrome (PCOS), another condition with underlying insulin resistance. The clinical applications of metformin are continuing to expand into other fields including cancer, aging, cardiovascular diseases, and neurodegenerative diseases. Metformin modulates multiple biological pathways. Its novel properties and effects continue to evolve; however, its molecular mechanism of action remains incompletely understood. In this chapter, we focus on the recent translational research and clinical data on the molecular action of metformin and the evidence linking the effects of metformin on insulin resistance, prediabetes, diabetes, aging, cancer, PCOS, cardiovascular diseases, and neurodegenerative diseases

Pancreatic islet transplantation to treat diabetes - defining molecular tools to select suitable islets [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionA complete understanding of pancreatic islet biology is essential to the development of preventive or curative interventions for diabetes. It has been known that subpopulations of islets of different sizes exist; however, whether they are biologically and functionally unique has not been investigated. As an example, our work comparing the biology of large versus small islets isolated from rats showed that small islets were superior to large islets in in vitro function and in transplantation outcomes. These results provided the stimulus for an improved approach to islet transplantation in humans. The work also led to new questions regarding the basic physiology of healthy islets. Through collaboration between our University of Kansas Medical Center and Children's Mercy Hospital teams, we determined that small islets secrete higher amount of insulin in vitro when compared to the large islets. We sought to identify whether the islet subpopulations showed differences at the molecular level and thus we investigated their protein expression profiles using two-dimensional polyacrylamide gel electrophoresis (2D PAGE). We found that the protein repertoire in the small and large islets differed significantly. Specifically, some proteins were found only in one type of islets, small or large, while they were missing or their expression levels were different in the other subpopulation. We identified some of the proteins by liquid chromatography - mass spectrometry. Immunofluorescence performed on small and large islets in pancreatic sections, with antibodies against identified proteins, confirmed that the proteins were present in one subpopulation of islets. Of these proteins, at least one was unique to large islets and can potentially be used as a marker to distinguish in vivo between islets that are high-insulin producers and those that fail to secrete significant amounts in insulin. Our long-term goal is to monitor the fate of the different islet populations during diabetes development. In addition, markers like this can be used to determine the best islet subpopulation for transplantation. The data support our hypothesis that integral differences exist between small and large islets that might determine the islets' unique properties under normal conditions and during the development of diabetes. These differences may also influence islet subpopulation behavior in transplantation affecting the outcome

Light Control of Insulin Release and Blood Glucose Using an Injectable Photoactivated Depot

In this work we demonstrate that blood glucose can be controlled remotely through light stimulated release of insulin from an injected cutaneous depot. Human insulin was tethered to an insoluble but injectable polymer via a linker, which was based on the light cleavable di-methoxy nitrophenyl ethyl (DMNPE) group. This material was injected into the skin of streptozotocin-treated diabetic rats. We observed insulin being released into the bloodstream after a 2 min trans-cutaneous irradiation of this site by a compact LED light source. Control animals treated with the same material, but in which light was blocked from the site, showed no release of insulin into the bloodstream. We also demonstrate that additional pulses of light from the light source result in additional pulses of insulin being absorbed into circulation. A significant reduction in blood glucose was then observed. Together, these results demonstrate the feasibility of using light to allow for the continuously variable control of insulin release. This in turn has the potential to allow for the tight control of blood glucose without the invasiveness of insulin pumps and cannulas

Expression and regulation of nampt in human islets.

Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD+ biosynthesis of a salvage pathway and exists in 2 known forms, intracellular Nampt (iNampt) and a secreted form, extracellular Nampt (eNampt). eNampt can generate an intermediate product, nicotinamide mononucleotide (NMN), which has been reported to support insulin secretion in pancreatic islets. Nampt has been reported to be expressed in the pancreas but islet specific expression has not been adequately defined. The aim of this study was to characterize Nampt expression, secretion and regulation by glucose in human islets. Gene and protein expression of Nampt was assessed in human pancreatic tissue and isolated islets by qRT-PCR and immunofluorescence/confocal imaging respectively. Variable amounts of Nampt mRNA were detected in pancreatic tissue and isolated islets. Immunofluorescence staining for Nampt was found in the exocrine and endocrine tissue of fetal pancreas. However, in adulthood, Nampt expression was localized predominantly in beta cells. Isolated human islets secreted increasing amounts of eNampt in response to high glucose (20 mM) in a static glucose-stimulated insulin secretion assay (GSIS). In addition to an increase in eNampt secretion, exposure to 20 mM glucose also increased Nampt mRNA levels but not protein content. The secretion of eNampt was attenuated by the addition of membrane depolarization inhibitors, diazoxide and nifedipine. Islet-secreted eNampt showed enzymatic activity in a reaction with increasing production of NAD+/NADH over time. In summary, we show that Nampt is expressed in both exocrine and endocrine tissue early in life but in adulthood expression is localized to endocrine tissue. Enzymatically active eNampt is secreted by human islets, is regulated by glucose and requires membrane depolarization

Protein levels of Nampt in human islets are greater with age.

<p>Examples of Nampt (blue) and insulin (green) immunofluorescence co-staining in islets from donors varying from 19 weeks gestation to 72 years old. <b>A–D:</b> In fetus and young children Nampt staining was weak with little co-localization with insulin in beta cells. <b>E–H:</b> In adults, Nampt staining was stronger and more localized to beta cells. <b>I:</b> Analysis of the Nampt pixel intensity illustrates the change with age.</p

Nampt in situ binding assay – summary of conditions tested.

<p>PFA, paraformaldehye; HCL, hydrochloric acid; RT, room temperature.</p

Co-localization of insulin, glucagon, and Nampt.

<p>Immunofluorescence image of an islet from an adult male stained for insulin, glucagon and Nampt. <b>A:</b> insulin staining (beta cells) using anti-insulin antibody (green). <b>B:</b> Glucagon was identified in the same islet (alpha cells) using anti-glucagon antibody (blue). <b>C:</b> Nampt was identified in the same islet using anti-Nampt antibody (red) and is found in both the islet and surrounding exocrine tissue. <b>D:</b> Overlap of all 3 images shows that the majority of Nampt co-localizes with insulin in beta cells.</p

Donor Demographics.

<p>KU Path, University of Kansas Pathology; BTB, National Institute of Childhood Diseases Brain and Tissue bank for Developmental Disorders at the University of Maryland, Baltimore MD; n/a, not available.</p

Protein pattern of Nampt in human islets changes with age.

<p>The difference in Nampt staining in endocrine and exocrine cells is clear. A: Fetal pancreatic tissues showed nearly equal Nampt staining levels in endocrine (within white circled regions) and exocrine tissue. B: In contrast, tissue from a 39 year old shows bright Nampt staining within the islet. C: The ratio of endocrine to exocrine pixel intensity illustrates the change with age. Of note, total image brightness was increased by 20% for every pancreatic image analyzed for figure C in order to visualize the low levels of Nampt staining in the fetal tissues.</p