Original Article Mechanisms of Glucose-Induced Secretion of Pancreatic-Derived Factor (PANDER or FAM3B) in Pancreatic -Cells Jichun Yang, Claudia E. Robert, Brant R. Burkhardt, Robert A. Young, Jianmei Wu, Zhiyong Gao, and Bryan A. Wolf Pancreatic-derived factor (PANDER) is an islet-specific cytokine present in both pancreatic - and -cells, which, in vitro, induces -cell apoptosis of primary islet and cell lines. In this study, we investigated whether PANDER is secreted by pancreatic - and -cells and whether PANDER secretion is regulated by glucose and other insulin secre- tagogues. In mouse-derived insulin-secreting -TC3 cells, PANDER secretion in the presence of stimulatory concen- trations of glucose was 2.8  0.4-fold higher (P < 0.05) than without glucose. Insulin secretion was similarly in- creased by glucose in the same cells. The total concentra- tion of secreted PANDER in the medium was 6 –10 ng/ml (0.3– 0.5 nmol/l) after a 24-h culture with glucose. L-Glu- cose failed to stimulate PANDER secretion in -TC3 cells. KCl stimulated PANDER secretion 2.1  0.1-fold compared with control without glucose. An L-type Ca 2 channel in- hibitor, nifedipine, completely blocked both glucose- or KCl-induced insulin and PANDER secretion. In rat-derived INS-1 cells, glucose (20 mmol/l) stimulated PANDER secre- tion 4.4  0.9-fold, while leucine plus glutamine stimulated 4.4  0.7-fold compared with control without glucose. In mouse islets overexpressing PANDER, glucose (20 mmol/l) stimulated PANDER secretion 3.2  0.5-fold (P < 0.05) compared with basal (3 mmol/l glucose). PANDER was also secreted by -TC3 cells but was not stimulated by glucose. Mutations of cysteine 229 or of cysteines 91 and 229 to serine, which may form one disulfide bond, and truncation of the COOH-terminus or NH 2 -terminus of PANDER all resulted in failure of PANDER secretion, even though these mutant or truncated PANDERs were highly expressed within the cells. In conclusion, we found that 1) PANDER is secreted from both pancreatic - and -cells, 2) glucose stimulates PANDER secretion dose dependently in -cell lines and primary islets but not in -cells, 3) PANDER is likely cosecreted with insulin via the same regulatory mechanisms, and 4) structure and conformation is vital for PANDER secretion. Diabetes 54:3217–3228, 2005 T ype 1 diabetes is an autoimmune disease result- ing from progressive destruction of insulin-se- creting -cells of pancreatic islets (1). It has been established that cytokines (mainly interleu- kin [IL]-1, tumor necrosis factor [TNF]-, and interferon [IFN]-) play vital roles in -cell dysfunction and death and in the development of type 1 diabetes (2– 6). Recently, it was shown that glucose causes islet -cells to produce IL-1, while the released IL-1 has a deleterious effect on human pancreatic islets (7,8). These studies suggest that production and release of cytokines from pancreatic islet cells are involved in -cell dysfunction and death in hyperglycemia. To date, although several cytokines have been shown to be involved in -cell dysfunction and death, the precise mechanisms of type 1 diabetes are still incom- pletely understood, suggesting that other potential factors may be involved. Pancreatic-derived factor (PANDER), also known as FAM3B (9,10), is one of four members of a new cytokine family recently identified using the algorithm ostensible recognition of folds (11) while searching for novel cyto- kines based on their predicted secondary structure. The rationale for this approach is that the secondary structure of cytokines is highly conserved through evolution. Many cytokines, such as IL-2, -3, -4, -5, -6, -7, -9, -10, and -13, and leptin share similar four-helix bundle secondary structures with a typical up-up-down-down typology (12,13). The PANDER gene encodes a 235–amino acid protein with a secretion signal peptide (aa1-29) and four cysteines (C63, C69, C91, and C229), which may form two putative disul- fide bridges (9). The PANDER gene is strongly expressed in pancreatic islets and slightly expressed in the prostate and small intestine. The tissue-specific expression implies that PANDER may regulate islet cell function as a locally produced cytokine. Our previous studies showed that PANDER protein is present in - and -cells of pancreatic islets. Furthermore, recombinant PANDER protein in- duces apoptosis of - and -cells of mouse, rat, and human islets in a dose- as well as time-dependent manner (10). Treatment with IFN- or a combination of IL-1, TNF-, and IFN- upregulates PANDER gene expression in mouse islets and in insulin secreting -cell lines in a time- and dose-dependent manner (14), revealing that PANDER may function as a downstream signal in cytokine-mediated -cell dysfunction and death. The PANDER promoter contains glucose-responsive elements and has a robust glucose response in pancreatic -cell lines and in primary From the Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. Address correspondence and reprint requests to Dr. Bryan A. Wolf, Depart- ment of Pathology and Laboratory Medicine, Children’s Hospital of Philadel- phia, 5135 Main Bldg, 34th St. and Civic Center Blvd., Philadelphia, PA 19104-4399. E-mail: wolfb@mail.med.upenn.edu. Received for publication 6 May 2005 and accepted in revised form 3 August 2005. CCH, carbachol; FBS, fetal bovine serum; IFN, interferon; IL, interleukin; KRBB, Krebs-Ringer bicarbonate buffer; PANDER, pancreatic-derived factor (or FAM3B); TBST, Tris-buffered saline with Tween; TNF, tumor necrosis factor. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DIABETES, VOL. 54, NOVEMBER 2005 3217