Pancreatic Beta-cell Tumors Research Articles

Exploring dual effects of dinutuximab beta on cell death and proliferation of insulinoma.

Insulinoma INS-1 cells are pancreatic beta cell tumors. Dinutuximab beta (DB) is a monoclonal antibody used in the treatment of neuroblastoma. The aim of this study is to investigate the effects of DB on pancreatic beta cell tumors at the molecular level. DB (Qarziba®) was available from EUSA Pharma. Streptozotocin (STZ) was used induce to cell cytotoxicity. DB was applied to the cells before or after the STZ application. KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were analyzed by q-RT-PCR, and protein levels were analyzed by Western blotting. Analysis of glucose-stimulated insulin secretion was performed. Ca+2 and CA19-9 levels were determined by the ELISA kit. PERK, CHOP, HSP90, p-c-Jun, p-Atf2, and p-Elk1 protein levels were analyzed by simple WES. Decreased KCND3, KCNK1, and PTHrP protein levels and increased KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were observed with DB applied after STZ application. Cell dysfunction was detected with DB applied before and after STZ application. Ca19-9 and Ca+2 levels were increased with DB applied after STZ application. PERK, CHOP, and p-Elk1 levels decreased, while HSP90 levels increased with DB applied after STZ application. CHOP, p-Akt-2, and p-c-Jun levels increased in the DB group. As a result, INS-1 cells go to cell death via the ERK signaling pathway without ER stress and release insulin with the decrease of K+ channels and an increase in Ca+2 levels with DB applied after STZ application. Moreover, the cells proliferate via JNK signaling with DB application. DB holds promise for the treatment of insulinoma. The study should be supported by in vivo studies.

BuOH fraction of Salix Babylonica L. extract increases pancreatic beta-cell tumor death at lower doses without harming their function

Salix babylonica L. is a species of the willow tree. Insulinoma is a tumor originating from pancreatic beta cells. This study aims to research the effect of different fractions of Salix babylonica L. leaf extract on INS-1 cells for treating pancreatic tumors. Cell death occurred at lower doses in the EtOAc fraction. The cells are functional in the BuOH fraction but not in EtOAc and H2O fractions. The EtOAc fraction has a higher percentage of necrosis and ROS. INS1, INS2, and AKT gene expressions in the H2O fraction, GLUT2, IR, HSP70 gene expressions, and WNT4 protein levels increased in the BuOH fraction. HSP90 gene expression, Beta-actin, GAPDH, insulin, HSP70, HSP90, HSF1, Beta-Catenin, and WNT7A protein levels were decreased, while IR immunolabelling intensity increased in both fractions. Ca+2, K+, Na+, and CA-19-9 in the cell, Ca+2 and K+ in secretion increased. The secondary metabolites in the EtOAc fraction cause more damage in INS-1 cells. Since the water fraction also causes the cells to die in high doses, cell function is damaged. The secondary metabolites in the BuOH fraction kill INS-1 cells with less damage. This makes the BuOH fraction of Salix babylonica L. more valuable.

Enucleation of Pancreatic Insulinomas with the use of Intraoperative Ultrasonography

Tumor-induced hypoglycaemia is very rare, usually caused by a pancreatic beta cell tumor (insulinoma). Symptoms caused by inappropriate insulin secretion usually occur during fasting periods, they can be neurogenic or neuroglycopenic. The diagnosis usually takes up to 2-years, this is biochemical with imaging support. The only curative treatment for an insulinoma is surgical resection (enucleation or partial pancreatectomy).

4-Methylcatechol stimulates apoptosis and reduces insulin secretion by decreasing betacellulin and inhibin beta-A in INS-1 beta-cells.

Insulinoma INS-1 cell line is a pancreatic beta cell tumor which is characterized with high insulin content and secretion in response to increasing glucose levels. 4-Methylcatechol (4-MC) is a metabolite of quercetin, which is known as a potential drug for inhibition of tumorigenesis. The aim of this study was to determine the applying doses of 4-methylcatechol (4-MC) for triggening cell death and decreasing the cell function of rat insulinoma INS-1 beta cells. The rate of apoptosis and the amount of insulin in the cell and the secretions were determined by the ELISA method. Betacellulin (BTC) and inhibin beta-A amounts in both the cell and the glucose induced secretion were investigated by Western blotting. Furthermore, BTC, Inhibin beta-A, Ins1, Ins2, and GLUT2 gene expression levels were determined by the by the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. We noted a significant decrease in cell viability, while an increase in apoptotic cell death by 4-MC treatment. It caused a decrease in the secretion of BTC, expressions of both BTC and inhibin beta-A. We showed a decrease in the expressions of Ins1 and GLUT2, while there is no alteration in the level of insulin protein. Insulin secretion levels increased in INS-1 cells given 4-MC by basal glucose concentration while they did not response to high concentration of glucose, which indicates that 4-MC disrupts the functionality of INS-1 cells. These results revealed that 4-MC induces apoptosis and decreases insulin secretion by reducing BTC and inhibin beta-A in insulinoma INS-1 cells. Thus, 4-MC may be offered as a potential molecule for treatment of insulinoma.

Abstract 478: A mouse model for highly metastatic, nonfunctioning pancreatic neuroendocrine tumors

Abstract Pancreatic neuroendocrine tumors are generally classified as functioning or nonfunctioning. Patients with functioning tumors can have severe hypoglycemia because their tumors express high amounts of insulin as well as chromogranin A; fortunately, these tumors are seldom metastatic. Patients with nonfunctioning tumors express high amounts of chromogranin A but little insulin; unfortunately these tumors are highly metastatic to the liver. Nonfunctioning tumors are more common, and a more severe health issue. The highly studied RT2 B6 mouse, in which the SV40 T antigen is expressed from an insulin promoter in an inbred C57Bl/6 genetic background, is a model for functioning pancreatic neuroendocrine tumors: liver metastasis is uncommon in RT2 B6 mice, which are very hypoglycemic and often die when tumors are very small. We have changed the genetic background and are studying RT2 AB6F1 hybrid mice, which have a C57Bl/6 father and a mother from an A/J inbred background. RT2 AB6F1 mice are much less hypoglycemic, and these mice develop liver metastasis with high frequency. We have mated the hybrid AB6F1 mice to their littermates in order to map metastasis genes by F2 analysis, and have discovered that a locus at mouse chromosome 2qG1 links tightly to liver metastasis. One gene within this locus, InsM1, is less expressed in the primary tumors of mice with liver metastatic disease. InsM1 is also less expressed in primary tumors from humans with liver metastatic disease. Previous studies of InsM1 have shown that this protein maintains the differentiated status of pancreatic beta cells. We find that tumors from mice with low expression of InsM1 also express lower amounts of several distinctive markers of differentiated beta cells, but higher amounts of several markers of liver cells. These data suggest that dedifferentiation to stem cells, which can then redifferentiate to other cell types such as liver cells, promotes metastasis of pancreatic beta cell tumors. Intratumoral expression of liver markers may be a useful predictor of metastatic disease in these patients. Dedifferentiation of mature beta cells is also a mechanism by which type 2 diabetes occurs, so there may be similarities between these two diseases. Citation Format: Tanupriya Contractor, Shinta Kobayashi, Richard Clausen, Edaise da Silva, Laura Tang, Chang Chan, Chris R. Harris. A mouse model for highly metastatic, nonfunctioning pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 478.

Diagnosis and treatment of an insulinoma in a guinea pig (Cavia porcellus)

A 5-year-old male guinea pig (Cavia porcellus) was examined because of lethargy, weight loss, and episodic neurologic signs, including paddling in lateral recumbency, head tilt, and circling. Prior to initial examination, the animal was treated with corn syrup whenever it appeared lethargic, plus an unknown dosage of sulfadimethoxazole. The animal was thin, with abdominal distention and subtle torticollis. Chemistry panel results documented hypoglycemia (45 mg/dL). Corn syrup was discontinued in favor of a high-fiber formula fed via a syringe. Measurement of blood insulin concentration demonstrated hyperinsulinemia (> 1,440 pmol/L [> 201 μU/L]), with concurrent hypoglycemia (0.6 mmol/L [11 mg/dL]). Diazoxide treatment for presumptive insulinoma was started at a dosage of 5 mg/kg (2.3 mg/lb), p.o., every 12 hours. A blood glucose curve demonstrated persistent hypoglycemia, and the diazoxide dosage was gradually increased to 25 mg/kg (11.4 mg/lb), p.o., every 12 hours. A second glucose curve measurement 12 days later confirmed adequate euglycemic control. Three weeks after the initial diazoxide dosage increase, the animal was reexamined for constipation and abdominal distension and died the following day. Histologic analysis confirmed a pancreatic beta-cell tumor (insulinoma). To the authors' knowledge, this is the first report of premortem diagnosis and treatment of an insulinoma in a guinea pig. This case demonstrates that diazoxide treatment can help achieve euglycemia in hypoglycemic guinea pigs and is a potential treatment option for guinea pigs with insulinoma.

Surgical Management of Adrenal Tumors and Insulinomas in Ferrets

Domestic ferrets are popular pets and comprise a significant percentage of the caseload in many veterinary practices. This article describes the diagnosis and surgical techniques for treatment of 2 common endocrine disorders affecting ferrets, adrenal gland disease and pancreatic beta cell neoplasms. Although medical treatment options are used to reduce the severity of clinical signs associated with adrenal hyperplasia or neoplasia, surgical removal of the adrenal gland(s) is the treatment of choice. Clinical disease associated with pancreatic beta cell tumors includes hypoglycemia, and in many cases the tumor will metastasize early in the course of the disease. Although achieving a complete resolution of pancreatic beta cell neoplasia is unlikely, surgical removal of insulin-secreting tumors is recommended to temporarily alleviate the clinical syndrome and confirm the diagnosis.

Ferret soft tissue surgery

Private veterinary practitioners are seeing pet ferrets with increasing frequency, and many common disease syndromes of the pet ferret require surgical intervention. This article discusses adrenal gland disease and adrenalectomy, prostatic and paraurethral cysts and their surgical management, perineal urethrostomy, pancreatic beta-cell tumor surgery, and splenectomy in the ferret. The surgical procedures are described with anatomic and physiologic information included when specific to the ferret. Indications for surgery, recomended presurgical diagnostics, and perioperative medical therapy are reviewed. Successful ferret soft tissue surgery depends on the surgeon's skill in combination with a working knowledge of ferret anatomy and the unique pathophysiology associated with the common ferret disease syndromes.

Auto-induction and growth stimulatory effect of betacellulin in human pancreatic cancer cells.

Betacellulin (BTC) was identified in mouse pancreatic beta cell tumors as a member of the epidermal growth factor (EGF) family, and was found to bind and activate the EGF receptor. BTC is also expressed in some human malignancies and may have an important role in tumor growth progression. We examined whether BTC and EGF have a growth stimulatory effect on human pancreatic cancer cell lines both in vitro and in vivo. We also investigated the BTC expression and autonomous induction of BTC in pancreatic cancer cells. in vitro, both BTC and EGF had almost the same proliferative effect on Panc-1, MIA PaCa-2 and AsPC-1. in vivo, in a Panc-1 inoculated athymic mice model, BTC-treated tumors grew approximately five times larger than in control. Immunocytochemistry showed that BTC expression occurred in three pancreatic cancer cell lines, with MIA PaCa-2 showing the strongest intensity. Semi-quantitative RT-PCR of MIA Paca-2 showed that mRNA levels of BTC gradually increased after treatment with 1 nM BTC. Immunocytochemistry also demonstrated that the intensity of BTC-like immunoreactivity was increased when treated with 1 nM BTC but was reduced after treatment with 100 nM of AG1478, an EGF receptor tyrosine kinase inhibitor. BTC has thus a significant growth stimulatory effect on pancreatic cancer cells and might function as an autocrine and paracrine growth factor. BTC expression in pancreatic cancer cells is, at least in part, controlled by an auto-induction mechanism.

Expression of IGF system genes during T-antigen driven pituitary tumorigenesis.

Expression of IGF-I, IGF-II, the Type-I IGF receptor and six IGF binding proteins were examined in three different T-ag-driven mouse tumors. Unlike the widespread expression of IGF-II in pancreatic beta-cell tumors, IGF-II was not widely expressed in the two different pituitary tumors examined indicating that a mechanism independent of focal IGF-II expression can also drive T-antigen tumorigenesis. In addition, multiple IGF binding proteins were expressed in all three tumor types. This expression, however, was generally heterogeneous with no specific changes to indicate a required role for any IGF binding protein in T-antigen tumorigenesis.

Pancreatic beta cell tumor in ferrets: 20 cases (1986–1994)

Objective— To characterize survival times, disease-free intervals, and prognostic variables in ferrets with pancreatic beta cell neoplasia. Design— Retrospective study. Animals— 20 ferrets with histologically confirmed pancreatic beta cell tumors. Procedure— Medical records of ferrets with pancreatic beta cell tumors were reviewed. Variables such as age, sex, duration of clinical signs, existence of other neoplasia, single versus multiple masses in the pancreas, benign or malignant histologic features, and the type of treatment used at redevelopment of clinical signs were examined to determine their relationship to survival times and disease-free intervals. Results— Duration of clinical signs prior to diagnosis and initial surgery was inversely correlated with survival time and disease-free interval. Overall median survival time for all ferrets with pancreatic beta cell tumors was 483 days. Median disease-free interval for ferrets that had abdominal surgery for pancreatic beta cell tumor removal was 240 days. Distant tumor metastasis was not found in this group of ferrets, but local tumor recurrence was common. In 4 ferrets that had a second celiotomy, removal of recurrent pancreatic tumors did not prolong survival but did result in a second disease-free interval. Clinical Implications— Pancreatic beta cell tumors should be considered in ferrets over 2 years of age that have clinical signs of seizures, episodic lethargy, ptyalism, ataxia, and hind limb weakness. In the study reported here, complete surgical excision of the tumor resulted in temporary resolution of clinical signs in ferrets; however, redevelopment of clinical signs at a later time was common. (J Am Vet Med Assoc 1996;209:1737–1740)

Glucose metabolism and insulin release in mouse beta HC9 cells, as model for wild-type pancreatic beta-cells.

Glucose metabolism and its relationship with glucose-induced insulin release were studied in beta HC9 and beta TC3 cells to identify and characterize key factors controlling the intermediary metabolism of glucose and glucose-induced insulin release. The beta HC9 cell line, derived from pancreatic islets with beta-cell hyperplasia, is characterized by a normal concentration-dependency curve for glucose-stimulated insulin release, whereas the beta TC3 cell line, derived from pancreatic beta-cell tumors, shows a marked leftward shift of this curve. Maximum velocity and the Michaelis-Menten constant of glucose uptake in beta HC9 and beta TC3 cells were similar, even though GLUT-2 expression in these two cell lines differed. In both cell lines, the kinetic characteristics of glucose usage, glucose oxidation, and glucose-induced oxygen consumption were similar to those of glucose phosphorylation, indicating that the kinetics of glucose metabolism from the glucose phosphorylation step in the cytosol to the mitochondrial process of oxidative phosphorylation are determined by the glucose-phosphorylating enzyme, that is, by glucokinase in beta HC9 cells and by hexokinase in beta TC3 cells. Thus beta HC9 cells provide an opportunity for the quantitative analysis of glucose metabolism, the associated generation of coupling factors, and other essential beta-cell functions involved in glucose sensing and insulin secretion.

Galanin Receptors in a Hamster Pancreatic β-Cell Tumor: Identification and Molecular Characterization

High affinity binding sites for galanin are identified and characterized in membranes from a hamster pancreatic beta-cell tumor. Using the radioiodinated peptide [125I] galanin, interaction of the peptide with pancreatic membranes is shown to be saturable, reversible, and time, temperature, membrane protein concentration, pH, and ionic strength dependent. In optimized equilibrium conditions of binding (90 min at 10 C), native galanin competitively inhibits the binding of [125I]galanin in a dose-dependent manner (from 10(-11)-10(-8) M); half-maximal inhibition is induced by 1 nM peptide. Scatchard analysis indicates the existence of a single population of sites of high affinity (Kd = 1.5 nM) and low capacity (44 fmol/mg protein). The monophasic dissociation process confirms the homogeneity of galanin-binding sites. Galanin-binding sites are highly specific, since apart from native galanin, none of the numerous biologically active peptides tested competes with [125I] galanin for binding to pancreatic membranes. The cross-linking of [125I]galanin to beta-cell membranes is performed using the chemical bifunctional reagent ethylene glycol bis-(succinimidyl succinate). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the presence or absence of dithiothreitol, one single band of 57,000 mol wt is observed, which may be corresponding to the [125I]galanin-receptor complex. Indeed, labeling of this 57,000 mol wt component is abolished only by native galanin but is unaffected by various other digestive peptides. Assuming one molecule of [125I]galanin is bound per molecule of protein, a 54,000 mol wt protein is identified as the pancreatic galanin receptor. In conclusion, our results indicate for the first time the identification of galanin receptors. Their presence in pancreatic beta-cells suggests a direct role of galanin in regulating endocrine beta-cell function.

Cell surface antigens on rat islet tumors.

To examine the antigenic properties of the rat pancreatic beta cell tumor, RIN, we used a cell surface enzyme-linked immunosorbent assay (CELISA). Monoclonal antibodies of known specificity are used to validate the assay, and the results show that antibodies directed at glycoproteins and glycolipids are detected by this technique. The principal glycolipid targets are found in the ganglio and lacto series of glycosphingolipids, and not in the globo series. When the CELISA was used to study sera from type I diabetics, no differences in binding of control and diabetic samples were detected at low dilutions of serum. However, at higher serum dilutions (1/30 to 1/120) binding of IgG antibodies from type I diabetics was greater than that observed with normal sera. Similar reactivity was not detected in these sera when rat hepatocytes were used as targets in the CELISA. Prolonged culture of RIN cells, however, resulted in the loss of reactivity with sera from type I subjects, and is associated with a corresponding decrease in expression of cell surface gangliosides. Accordingly, subclones selected for ganglioside expression were used to compare anti-RIN binding in type I diabetics with that of normal controls and unaffected siblings. The results indicate that some rat islet tumors express antigenic determinants recognized by anti-islet antibodies associated with type I diabetes. Both nonspecific interaction at low serum dilutions and variable expression of cell surface antigens may explain the difficulties encountered when these cells are used for diagnostic purposes. An objective assay such as the CELISA may help to avoid these problems.

The CIP receptor on pancreatic beta cell tumor: Molecular identification by covalent cross-linking

125I-GIP binds reversibly to a high affinity binding site in crude plasma membranes prepared from a hamster pancreatic β cell tumor. The treatment of labeled membranes with the cross-linker dithiobis (succinimidylpropionate) prevents, to a great extent, the rapid dissociation of 125I-GIP-membrane complexes which is observed when 10 −6M native GIP is added. Polyacrylamide gel electrophoresis of membrane proteins reveals a major 125I-GIP-protein complex of Mr 64,000. This labeling decreases when increasing concentrations (10 −9–10 −6M) of native GIP are added but is not altered by other peptide hormones (tested at 10 −6M) including glucagon, VIP and insulin. The Mr 64,000 complex is not observed in tissues which have no specific binding sites for GIP such as intestinal epithelium. Assuming one molecule of 125I-GIP is bound per molecule of protein, one protein with Mr 59,000 is identified as the specific GIP binding site.

Feedback-controlled dextrose infusion during surgical management of insulinomas

Through the use of a feedback-controlled dextrose infusion system, we obtained continuous monitoring of the blood glucose level in a patient undergoing surgery for multiple pancreatic beta-cell tumors. In addition, this device infused dextrose at variable rates to maintain a predetermined euglycemic level of 90 mg/dl. Before locating the source of excessive insulin production, the maximum dextrose infusion rate of 400 mg/min was required; but after extirpation of multiple insulinomas, the dextrose-infusion rate declined whereas the blood glucose level rose above the preselected level These results emphasize the usefulness of a monitoring and infusion system not only in protecting the patient from the hazard of hypoglycemia under anesthesia but also as an aid in determining whether all insulin-secreting tumors have been removed.

Responses of Patients with Insulinomas to Stimulators and Inhibitors of Insulin Release That Have Been Linked with Cyclic Adenosine Monophosphate

The study comprises nine consecutive patients who had pancreatic beta-cell tumors and spontaneous hypoglycemia, seven of whom had verified, benign insulinomas and one a malignant insulinoma with metastases to the liver. The ninth patient, who refused surgery, was followed for three years without displaying any signs of malignancy. Among the patients with benign insulinomas, three showed exaggerated, two normal, and two unusual insulin responses to glucose. One such patient as well as the one with a malignant tumor did not respond to glucose. In general, the insulin responses to glucagon were similar to those to glucose. Somatostatin reduced basal insulin levels as well as the responses to glucose and glucagon only in subjects with normal or exaggerated responses to these agents. Einephrine behaved like somatostatin when insulin release was stimulated by glucose, but it was a weak inhibitor of the glucagon-stimulated response. The subject with the malignant insulinoma did not respond to either glucose or somatostatin. The remission of the disease, which followed streptozotocin treatment, was accompanied by restoration of the normal responsiveness to the above agents. Glucagon stimulates insulin release by increasing the intracellular level of cyclic adenosine monophosphate (AMP). The effects of glucose, somatostatin, and epinephrine are supposed to be mediated, at least partially, by way of the adenylcyclase-cyclic AMP system. Therefore, the present data suggest that the variability of the beta-cell responsiveness to glucose and glucagon in patients with insulinoma reflects the responsiveness of the adenylcyclase-cyclic AMP system of the beta-cell tumor, which may be exaggerated, normal, or absent.

Spontaneous Pancreatic Beta Cell Tumor in the Rat

Fine structural study of a pancreatic neoplasm found at autopsy of a 27-month-old female Long-Evans rat showed that the tumor consisted of, and derived from beta cells of the islets of Langerhans.

FAILURE OF SOMATOSTATIN TO INHIBIT TOLBUTAMIDE-INDUCED INSULIN SECRETION IN PATIENTS WITH INSULINOMAS: A POSSIBLE DIAGNOSTIC TOOL

The effects of somatostatin on tolbutamide-stimulated insulin release were studied in 4 patients with insulin-producing tumors of the pancreas and in 6 normal subjects. In contrast to its effective inhibition of insulin release in normal subjects, somatostatin, without exception, failed to inhibit tolbutamide-induced insulin release in the patients with pancreatic beta-cell tumors. This differential effect of somatostatin may prove useful in the diagnosis of insulin-producing tumors of the pancreas.