Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells

Cells for therapeutic use are often preserved at +4 °C, and the storage period is generally limited to 2–3 days. Here, we report that the survival rate (%) of mammalian cells is improved to 10–20 days when they are preserved with a subzero supercooled solution containing the antifreeze protein (AFP), for which an ability to stabilize both supercooled water and cell membrane integrity has been postulated. We chose adherent rat insulinoma (RIN-5F) cells as the preservation target, which were immersed into −5 °C-, −2 °C-, or +4 °C-chilled “unfrozen” solution of Euro-Collins or University of Washington (UW) containing the AFP sample obtained from insect or fish. Our results show that the survival rate of the cells preserved with the solution containing insect AFP was always higher than that of the fish AFP solution. A combination of the −5 °C-supercooling and insect AFP gave the best preservation result, namely, UW solution containing insect AFP kept 53% of the cells alive, even after 20 days of preservation at −5 °C. The insect AFP locates highly organized ice-like waters on its molecular surface. Such waters may bind to semiclathrate waters constructing both embryonic ice crystals and a membrane–water interface in the supercooled solution, thereby protecting the cells from damage due to chilling.

Isolation and Proteomics of the Insulin Secretory Granule

Insulin, a vital hormone for glucose homeostasis is produced by pancreatic beta-cells and when secreted, stimulates the uptake and storage of glucose from the blood. In the pancreas, insulin is stored in vesicles termed insulin secretory granules (ISGs). In Type 2 diabetes (T2D), defects in insulin action results in peripheral insulin resistance and beta-cell compensation, ultimately leading to dysfunctional ISG production and secretion. ISGs are functionally dynamic and many proteins present either on the membrane or in the lumen of the ISG may modulate and affect different stages of ISG trafficking and secretion. Previously, studies have identified few ISG proteins and more recently, proteomics analyses of purified ISGs have uncovered potential novel ISG proteins. This review summarizes the proteins identified in the current ISG proteomes from rat insulinoma INS-1 and INS-1E cell lines. Here, we also discuss techniques of ISG isolation and purification, its challenges and potential future directions.

FoxA2 and RNA Pol II Mediate Human Islet Amyloid Polypeptide Turnover in ER-stressed Pancreatic β-cells

Here, we investigated transcriptional and trafficking mechanisms of human islet amyloid polypeptide (hIAPP) in normal and stressed β-cells. In high glucose-challenged human islets and rat insulinoma cells overexpressing hIAPP, cell fractionation studies revealed increased accumulation of hIAPP. Unexpectedly, a significant fraction (up to 22%) of hIAPP was found in the nuclear soluble and chromatin-enriched fractions of cultured human islet and rat insulinoma cells. The nucleolar accumulation of monomeric forms of hIAPP did not have any adverse effect on the proliferation of β-cells nor did it affect nucleolar organization or function. However, intact nucleolar organization and function were essential for hIAPP expression under normal and ER-stress conditions as RNA polymerase II inhibitor, α-amanitin, reduced hIAPP protein expression evoked by high glucose and thapsigargin. Promoter activity studies revealed the essential role of transcription factor FoxA2 in hIAPP promoter activation in ER-stressed β-cells. Transcriptome and secretory studies demonstrate that the biosynthetic and secretory capacity of islet β-cells was preserved during ER stress. Thus, the main reason for increased intracellular hIAPP accumulation is its enhanced biosynthesis under these adverse conditions.

Nanomolar Melatonin Influences Insulin Synthesis and Secretion in Rat Insulinoma INS-1E Cells

Background: Pancreatic beta-cell dysfunction results in reductions of insulin synthesis/secretion, cell survival, and insulin sensitivity thereby inducing diabetes mellitus. In this study, how nanomolar melatonin regulates insulin synthesis and secretion in rat insulinoma INS-1E cells was investigated. Methods: Western Blot and ELISA were performed to investigate the insulin synthesis and secretion at melatonin concentrations of 10-100 nM for 48 h and 10-500 nM for 72 h in rat insulinoma INS-1E cells.Results: Melatonin significantly increased the insulin protein level in INS-1E cells above the level in control cells without melatonin or glucose treatments and decreased the insulin protein level in media with glucose. At melatonin concentrations of 10-100 nM for 48 h and 10-500 nM for 72 h, increases and decreases occurred in dose-dependent manners. Luzindole or 4-phenyl-2-propionamidotetralin (4P-PDOT), melatonin receptor (MR) antagonists, inhibited the melatonin-induced insulin protein level in cells and media. Levels of membrane vesicle trafficking-related proteins including Rab5, GOPC, and phospho-caveolin-1 proteins significantly increased with melatonin treatment above that in control cells without melatonin or glucose treatments, whereas expressions of EEA1, clathrin, APPL1, and syntaxin-6 proteins significantly decreased with melatonin treatment. The increases in the phosphorylation of the mammalian target of rapamycin (p-mTOR) and Raptor protein levels (mTOR Complex 1 (mTORC1)) were consistent with the increments in the expressions of p-Akt (Ser473, Thr308). Also, expression levels of Bcl-2 and Bcl-xL proteins were significantly increased compared to those in control cells without melatonin or glucose treatments, whereas the Bax protein level decreased. Luzindole or 4P-PDOT decreased the melatonin-induced levels of p-mTOR (Ser2448), Raptor, p-Akt (Ser473), and Bcl-2 proteins, whereas MR antagonists increased the melatonin-suppressed level of Bax proteins. Conclusion: These results indicate that nanomolar melatonin regulates insulin synthesis and secretion associated with membrane vesicle trafficking-related proteins, including Rab5, GOPC, p-Caveolin-1, EEA1, and clathrin through the Akt/mTORC1 pathway.

Effects of Sclerocarya birrea Stem-Bark Extracts on Glucose Uptake, Insulin Synthesis and Expression of Selected Genes Involved in the Synthesis and Secretion of Insulin in Rat Insulinoma Pancreatic Beta Cells

The present study reported the effects of Sclerocarya birrea stem-bark (SBSB) extracts on glucose uptake, insulin synthesis and the expression of glucose transporter 2 (GLUT2), glucokinase, pancreatic duodenal homeobox-1 (PDX-1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) and pre-proinsulin genes in rat insulinoma (RIN)-m5F pancreatic beta cells. The amount of glucose takenup by RIN-m5F cells was measured using a glucose oxidase-based assay kit. Intracellular and secreted insulin were measured using an enzyme linked immunoassay kit. Pre-proinsulin gene expression was determined using the conventional polymerase chain reaction (PCR) technique, while the expressions of GLUT2, glucokinase, PDX-1 and MafA genes were evaluated using quantitative real-time PCR technique. Of the four SBSB extracts investigated in the study, only the SBSB hexane extract positively affected all the study variables in RIN-m5F cells compared with the DMSO control. Thus, the SBSB hexane extract contains phytochemicals capable of enhancing insulin synthesis partly through up-regulation of the expression of GLUT2, glucokinase, PDX-1, MafA and pre-proinsulin genes.

Identification of a small molecule that stimulates human β-cell proliferation and insulin secretion, and protects against cytotoxic stress in rat insulinoma cells

A key event in the development of both major forms of diabetes is the loss of functional pancreatic islet β-cell mass. Strategies aimed at enhancing β-cell regeneration have long been pursued, but methods for reliably inducing human β-cell proliferation with full retention of key functions such as glucose-stimulated insulin secretion (GSIS) are still very limited. We have previously reported that overexpression of the homeobox transcription factor Nkx6.1 stimulates β-cell proliferation, while also enhancing GSIS and providing protection against β-cell cytotoxicity through induction of the VGF prohormone. We developed an Nkx6.1 pathway screen by stably transfecting 832/13 rat insulinoma cells with a VGF promoter-luciferase reporter construct, using the resultant cell line to screen a 630,000 compound chemical library. We isolated three compounds with consistent effects to stimulate human islet cell proliferation. Further studies of the most potent of these compounds, GNF-9228, revealed that it selectively activates human β-cell relative to α-cell proliferation and has no effect on δ-cell replication. In addition, pre-treatment, but not short term exposure of human islets to GNF-9228 enhances GSIS. GNF-9228 also protects 832/13 insulinoma cells against ER stress- and inflammatory cytokine-induced cytotoxicity. In contrast to recently emergent Dyrk1a inhibitors that stimulate human islet cell proliferation, GNF-9228 does not activate NFAT translocation. These studies have led to identification of a small molecule with pleiotropic positive effects on islet biology, including stimulation of human β-cell proliferation and insulin secretion, and protection against multiple agents of cytotoxic stress.