Sagar, Gunisha

Background and objectives New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. Design Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. Results In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. Conclusions PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.

Abstract Purpose: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer–derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. Experimental Methods: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Results: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Conclusions: Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin+/CA19-9+ exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. Clin Cancer Res; 21(7); 1722–33. ©2014 AACR. See related commentary by Korc, p. 1508

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0–75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR−/− mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.