The combination of GIP plus xenin-25 indirectly increases pancreatic polypeptide release in humans with and without type 2 diabetes mellitus

Abstract

Xenin-25 (Xen) is a 25-amino acid neurotensin-related peptide that activates neurotensin receptor-1 (NTSR1). We previously showed that Xen increases the effect of glucose-dependent insulinotropic polypeptide (GIP) on insulin release 1) in hyperglycemic mice via a cholinergic relay in the periphery independent from the central nervous system and 2) in humans with normal or impaired glucose tolerance, but not type 2 diabetes mellitus (T2DM). Since this blunted response to Xen defines a novel defect in T2DM, it is important to understand how Xen regulates islet physiology. On separate visits, subjects received intravenous graded glucose infusions with vehicle, GIP, Xen, or GIP plus Xen. The pancreatic polypeptide response was used as an indirect measure of cholinergic input to islets. The graded glucose infusion itself had little effect on the pancreatic polypeptide response whereas administration of Xen equally increased the pancreatic polypeptide response in humans with normal glucose tolerance, impaired glucose tolerance, and T2DM. The pancreatic polypeptide response to Xen was similarly amplified by GIP in all 3 groups. Antibody staining of human pancreas showed that NTSR1 is not detectable on islet endocrine cells, sympathetic neurons, blood vessels, or endothelial cells but is expressed at high levels on PGP9.5-positive axons in the exocrine tissue and at low levels on ductal epithelial cells. PGP9.5 positive nerve fibers contacting beta cells in the islet periphery were also observed. Thus, a neural relay, potentially involving muscarinic acetylcholine receptors, indirectly increases the effects of Xen on pancreatic polypeptide release in humans.

Keywords: Alb; Cholinergic; G+X; GIP; GIP plus Xen; GLP-1; IGT; ISR; NGT; NTSR1; Neuronal relay; Neurotensin receptor; PP; PPR; Pancreatic polypeptide; T2DM; Xen; Xenin; albumin alone; glucagon-like peptide-1; glucose-dependent insulinotropic polypeptide; impaired glucose tolerance; insulin secretion rate; intravenous graded glucose infusions; ivGGIs; neurotensin receptor-1; normal glucose tolerance; pancreatic polypeptide; pancreatic polypeptide response; type 2 diabetes mellitus; xenin-25.

Conflict of interest statement

Conflicts of interest

Washington University is pursuing a patent related to the use of xenin-25 to treat T2DM. In the future, this could lead to personal financial benefit to Burton M. Wice and the University. No other potential conflicts of interest to this article were reported.

© 2013.

Figures

Figure 1. Fasting PP levels are similar in humans with NGT, IGT, and T2DM

Panel A. Amino acid sequences for neurotensin-related peptides are shown. Note that the amino termini of neurotensin and xenopsin (<Q) contain a cyclic modification of glutamine. Panel B. Following a 10-hour overnight fast, subjects were administered ivGGIs at the indicated glucose and peptide infusion rates. Panel C. Plasma glucose levels were measured at the indicated times during the ivGGIs without peptide administration. Group mean ± SEM for each time point is shown for subjects with NGT (green circles), IGT (yellow squares), and T2DM (red triangles). Data are taken from ref . Panel D. Changes in PP levels from 0 to 40 minutes of the ivGGI were measured in EDTA and heparinized plasma during infusion with Alb, GIP, Xen, or the combination of G+X. Values measured in the heparin versus EDTA samples from 25 subjects are shown. Panel E. PP standards were prepared in buffer supplied by the manufacturer (black circles) or charcoal-stripped heparinized (red squares) or EDTA (green triangles) plasma. Only the plasma samples were extracted with acetonitrile before PP measurements. Measured values are plotted versus the theoretical concentration in the diluted standards. Panel F. Fasting PP levels were measured in heparinized plasma samples extracted with acetonitrile. Average PP levels ± SEM are shown for each group.

Figure 2. The combination of GIP plus Xen similarly increases the PP response in humans with NGT, IGT, and T2DM

ivGGIs were administered as outlined in Fig 1A during infusion of Alb(yellow triangles), GIP (red squares), Xen (green inverted triangles), or G+X (blue circles). The PPR for the NGT, IGT, and T2DM groups at the indicated times are shown in panels A, D, and G. iAUCs for the PPR were calculated for each infusion and for each individual. Group average iAUC ± SEM over the first 40 minutes (panels B, E, and H) and the entire 240 minute ivGGI (panels C, F, and I) are shown. Note that unextracted plasma from some subjects contained compounds that interfered with PP measurements and their data was not included.

Figure 3. NSTR1 is not detectable on islet endocrine cells

Panels A–C: A single paraffin-embedded section of normal human pancreas was incubated with antibodies to NTSR1. To avoid potential artifacts due to autofluorescence of endocrine cells, bound primary antibodies were visualized using horseradish peroxidase conjugated secondary antibodies followed by incubation with diaminobenzidine. Nuclei were counterstained with hematoxylin. An intensely labeled axon (Panel A), a low expressing duct (Panel B), and an islet with undetectable levels of NTSR1 (Panel C) are shown.

Figure 4. NSTR1 is expressed on neurons in the pancreas

Panels A–D: A single paraffin-embedded section of normal human pancreas was incubated with antibodies to NTSR1 plus antibodies to PGP9.5. Nuclei were counterstained blue using bis-benzimide (Panel A). Bound primary antibodies were visualized using ALEXA 549 (Panel B; red) and ALEXA 488 (Panel C; green) secondary antibodies. Panel D is a merged image of Panels A–C. The open and solid arrows indicate PGP9.5 labeled axons that do and do not co-express NTSR1, respectively.

Figure 5. Neurons contact human islets

Panels A–E: A single paraffin-embedded section of normal human pancreas was stained with antibodies to PGP9.5 plus antibodies to insulin as described in Fig 4. Panels A, B, and C show staining only for nuclei (blue), insulin (green), and PGP9.5 (red), respectively. Panel D is a merged image of Panels A–C. Panel E is enlarged from the boxed region in panel D. Open arrows point to representative PGP9.5 positive nerve fibers. The closed arrow points to a nerve fiber contacting a double-labeled beta cell.

Figure 6. NTSR1 is not detectable on alpha cells

A single paraffin-embedded section of normal human pancreas was stained with antibodies to insulin plus antibodies to PGP9.5 as described in Fig 4. Panels A, B, and C show staining only for nuclei (blue), insulin (green), and PGP9.5 (red), respectively. Panel D is a merged image of Panels A–C. Panel E is enlarged from the boxed region in panel D. The open and closed arrows point to representative axons and alpha cells, respectively.

Figure 7. NTSR1 is not detectable on sympathetic neurons

A single paraffin-embedded section of normal human pancreas was stained with antibodies to tyrosine hydroxylase (TH) plus antibodies to NTSR1 as described in Fig 4. Panels A, B, and C show staining only for nuclei (blue), tyrosine hydroxylase (green), and NTSR1 (red), respectively. Panel D is a merged image of Panels A–C.