Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice

Abstract

Background & aims: New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer.

Methods: Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls).

Results: Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not.

Conclusions: Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice.

Figure 1

Analysis of gene expression PaC cell lines led to the identification of AM, a candidate mediator of the diabetogenic effect of these cell lines. (A) Effect of PaC cell supernatants on insulin secretion in INS1 cells showed inhibition of insulin secretion with supernatants from PANC1, L3.6, HPAFII, and SU86.86 as compared with HPDE6 cells. Data pooled from at least 3 experiments with each condition in triplicate. Error bars represent the SEM. (B) Microarray data using the Affymetrix Human U133 Plus 2.0 microarray in PaC cell lines showed a 7-fold overexpression of AM in PANC1 cells and to a lesser degree in SU86.86 compared with HPDE6 cells.

Figure 2

AM inhibits insulin secretion and contributes to the insulin inhibitory effect of PaC cells. (A) AM inhibits glucose-stimulated (15 mmol/L) insulin secretion in INS1 cells. (B) AM knockdown using AM shRNA ameliorates the inhibitory effect of PANC1 supernatants on insulin secretion in INS1 cells as compared with scramble (Scr) shRNA. (C) AM inhibits glucose-stimulated (16.7 mmol/L) insulin secretion in isolated mouse islets in a dose-dependent manner. (D) AM knockdown using AM shRNA ameliorates the inhibitory effect of PANC1 supernatants on insulin secretion in isolated mouse islets as compared with scramble (Scr) shRNA. Error bars represent the SEM.

Figure 3

AM overexpression in PaC in vivo leads to the development of glucose intolerance. (A) MPanc96 luciferase cells were injected subcutaneously and orthotopically in athymic nude mice (n = 10) and pancreatic tumor was developed. Wild-type mice served as control. By bioluminescence imaging, tumors were size matched, mice were subjected to glucose tolerance tests, and blood glucose levels were measured using a glucometer at different time points. Mice bearing both subcutaneous and orthotopic tumors showed a significant increase (P < .05) in blood glucose levels as compared with control mice at 15, 30, 60, and 90 minutes. (B) Orthotopic tumors were developed with and without CHO luciferase cells expressing AM in SCID mice (n = 10). By bioluminescence imaging, tumors were size matched, and mice were subjected to glucose tolerance tests and blood glucose levels were measured using a glucometer at different time points. Mice bearing tumors expressing AM showed a significant increase (P < .05) in blood glucose levels as compared with control tumor-bearing mice at 30, 45, 60, and 90 minutes.

Figure 4

AM is overexpressed in human PaC, especially in patients with DM, compared with control patients with and without other pancreatic diseases. (A) Immunohistochemistry for AM. (i) PaC with perineural invasion showing diffuse cytoplasmic staining (red arrow). Normal peritumoral acini (black arrow) and peritumoral islet (blue arrow) also show intense staining. (ii) AM stain shows darker, membranous staining in normal ducts (black arrow) whereas PaC has diffuse, cytoplasmic staining (red arrow). (iii and iv) In normal pancreas, only islets stain for AM (black arrows). (B) Quantitative RT-PCR on pancreatic tissue from human PaC tissue and controls shows marked AM overexpression in PaC compared with controls. (C) Quantitative RT-PCR on RNA isolated from human PaC tissue shows higher AM expression in patients with DM compared with patients without DM.

Figure 5

AM as a candidate biomarker of PaCDM. Fasting plasma AM levels (measured using radioimmunoassay) in 61 patients with PaC (31 with normal fasting glucose levels and 30 with new-onset type 2 DM) and 55 age- and sex-matched controls (28 patients with normal fasting glucose levels and 27 with new-onset type 2 DM). Mean AM levels are higher in patients with PaCDM (22.9 ± 10.7) compared with patients with PaC with normal fasting glucose levels (18.3 ± 7.0, P = .057), noncancer subjects with new-onset type 2 DM (14.8 ± 10.7; P < .001), and noncancer subjects with normal fasting glucose levels (12.9 ± 6.6; P < .001).