Insulinoma-derived pseudo-islets for diabetes research

The islets of Langerhans of the pancreas are the primary endocrine organ responsible for regulating whole body glucose homeostasis. The use of isolated primary islets for research development and training requires organ resection, careful digestion and isolation of the islets from non-endocrine tissue. This process is time consuming, expensive and requires substantial expertise. For these reasons, we sought to develop a more rapidly obtainable and consistent model system with characteristic islet morphology and function that could be employed to train personnel and better inform experiments prior to using isolated rodent and human islets. Immortalized β cell lines reflect several aspects of primary β cells, but cell propagation in monolayer cell culture limits their usefulness in several areas of research which depend on islet morphology and/or functional assessment. In this manuscript we describe the propagation and characterization of insulinoma pseudo-islets (IPIs) from a rat insulinoma cell line INS832/3. IPIs were generated with an average diameter of 200 μm, consistent with general islet morphology. The rates of oxygen consumption and mitochondrial oxidation-reduction changes in response to glucose and metabolic modulators were similar to isolated rat islets. In addition, the dynamic insulin secretory patterns of IPIs were similar to primary rat islets. Thus, INS832/3-derived IPIs provide a valuable and convenient model for accelerating islet and diabetes research.

Wild-type menin is rapidly degraded via the ubiquitin-proteasome pathway in a rat insulinoma cell line

Menin is encoded by multiple endocrine neoplasia type 1 (MEN1) gene, the germ line mutations of which are the main cause of pancreatic neuroendocrine tumors (PNETs). To date, a large number of frameshift, nonsense and missense mutations of MEN1 have been identified to be responsible for part of MEN1-defficient PNETs patients due to truncation or rapid degradation of menin protein. However, the stability of the wild-type (WT) menin in PNETs is totally unknown. In the present study, we observed ubiquitination of WT menin in 293T cells by transfection of ectopic WT menin and HA-ubiquitin. As expected, either endogenous or ectopic WT menin is stable in 293T cells, whereas in INS-1 cells, a rat insulinoma cell line derived from PNETs, either endogenous or ectopic WT menin is rapidly degraded through ubiquitin-proteasome pathway. Furthermore, the degradation of WT menin is more rapid in the presence of serum. Our findings suggest that in part of PNETs patients with WT MEN1, a ubiquitin-proteasome system targeting menin is untimely activated.

P38 Plays an Important Role in Glucolipotoxicity-Induced Apoptosis in INS-1 Cells

Objectives. The mechanism underlying the regulation of glucolipotoxicity-induced apoptosis by MAPKs was examined in INS-1 cells.Methods. The rat insulinoma cell line INS-1 was cotreated with glucose (30 mM) and palmitic acid (0.2 mM) (GLU+PA). Apoptosis was assessed by cell morphology and detection of PARP cleavage. The activation of MAPKs was examined by Western blotting using specific antibodies against the phosphorylated forms of JNK, ERK1/2, and P38.Results. (1) Live cell imaging studies showed that treatment with GLU+PA for 72 h induced significant cell death, concomitant with PARP-1 cleavage and caspase-3 activation, which peaked at 96 h of treatment. (2) Western blot analysis of the activation of MAPKs during GLU+PA-induced INS-1 cell apoptosis showed that phosphorylation of P38 increased gradually and reached a peak at 96 h, which coincided with PARP-1 cleavage. A transient increase of ERK activation was followed by a rapid decline at 96 h, whereas JNK phosphorylation status remained unchanged in response to GLU+PA. (3) Phosphorylation of insulin receptor substrate (IRS)-2 at 48 h of treatment triggered its degradation, which coincided with P38 activation. (4) Inhibition of P38, but not JNK or ERK, blocked GLU+PA-induced INS-1 cell apoptosis.Conclusions. P38 may be involved in the regulation of glucolipotoxicity-induced apoptosis through the phosphorylation of IRS-2.

Sp1/Sp3 binding is associated with cell-specific expression of the glucose-dependent insulinotropic polypeptide receptor gene

The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor ( GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between −1 and −100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5′-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between −85 and −40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions −77, −60, and −50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at −50 and −60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at −70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between −100 and −40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.