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

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

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

Quantification of Nampt mRNA

<p>. Total RNA was isolated from <b>A.</b> human pancreatic tissue or <b>B.</b> isolated human islets. NAMPT mRNA was quantified using a Nampt specific Taqman assay (Applied Biosystems/Life Technologies, Carlsbad, CA.) according to the manufacturer's instructions and normalized with GUSB. *Note: The Y axis uses C_T values (C_T is the threshold cycle of detection), thus increased target mRNA results in earlier detection by qRT-PCR (i.e., a smaller C_T).</p

The effects of glucose on Nampt gene and protein expression levels in human islets.

<p>Total RNA or protein was isolated from islets treated for 1 hour in 20 mM glucose. <b>A:</b> Nampt gene expression was upregulated in the presence of 20 mM glucose compared to control (2.2 mM glucose) by qRT-PCR. Each bar represents the mean fold change normalized to β-actin from five separate experiments.*P<0.05; student T test <b>B:</b> Nampt protein content did not increase in the presence of 20 mM compared to 2.2 mM glucose by western blot analysis. Shown is a representative blot from 6 separate experiments.</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

Nampt in situ binding assay – summary of conditions tested.

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