Decrease in glucose-stimulated insulin secretion following exposure to magnetic fields

We evaluated the effects of extremely low frequency magnetic field (ELFMF) on glucose-stimulated insulin secretion from HIT-T15 cells and investigated the mechanisms of these effects. We demonstrated that exposure to ELFMF at 5mT decreased glucose-stimulated insulin secretion by preventing the increases in cellular adenosine 5'-triphosphate/adenosine 5'-diphosphate, membrane depolarization, and cytosolic free calcium ion concentration. The glucose-induced upregulation of insulin mRNA expression was also attenuated by exposure to ELFMF, although cell viability was not affected. These findings demonstrate the potential of exposure to ELFMF for clinical use as a novel inhibitory method of insulin secretion.     

An extremely low frequency magnetic field attenuates insulin secretion from the insulinoma cell line, RIN-m

In this study, we investigated the effects of exposure to an extremely low frequency magnetic field (ELFMF) on hormone secretion from an islet derived insulinoma cell line, RIN-m. We stimulated RIN-m cells to secrete insulin under exposure to an ELFMF, using our established system for the exposure of cultured cells to an ELFMF at 5 mT and 60 Hz, or under sham exposure conditions for 1 h and observed the effects. In the presence of a depolarizing concentration of potassium (45 mM KCl), exposure to ELFMF significantly attenuated insulin release from RIN-m cells, compared to sham exposed cells. Treatment with nifedipine reduced the difference in insulin secretion between cells exposed to an ELFMF and sham exposed cells. The expression of mRNA encoding synaptosomal associated protein of 25 kDa (SNAP-25) and synaptotagmin 1, which play a role in exocytosis in hormone secretion and influx of calcium ions, decreased with exposure to an ELFMF in the presence of 45 mM KCl. These results suggest that exposure to ELFMF attenuates insulin secretion from RIN-m cells by affecting calcium influx through calcium channels.     

Effects of strong static magnetic fields used in magnetic resonance imaging on insulin-secreting cells

The magnetic flux density of MRI for clinical diagnosis has been steadily increasing. However, there remains very little biological data regarding the effect of strong static magnetic fields (SMFs) on human health. To evaluate the effects of strong SMFs on biological systems, we cultured insulin-secreting cells under exposure to sham and SMF conditions (3-10 T of magnetic flux density, and 0-41.7 T/m of magnetic field gradient) for 0.5 or 1 h, and analyzed insulin secretion, mRNA expression, glucose-stimulated insulin secretion, insulin content, cell proliferation and cell number. Exposure to SMF with a high magnetic field gradient for 1 h significantly increased insulin secretion and insulin 1 mRNA expression. Exposure to SMF with a high magnetic flux density for 0.5 h significantly enhanced responsiveness to glucose stimulation. Exposure to SMF did not affect the insulin content, cell proliferation or cell number. Our results suggested that MRI systems with a higher magnetic flux density might not cause cell proliferative or functional damages on insulin-secreting cells, and that SMF with a high magnetic field gradient might be used clinically after thorough in vivo investigations are conducted.     

Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.     

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca²⁺ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca²⁺ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Oxidized but not acetylated low-density lipoprotein reduces preproinsulin mRNA expression and secretion of insulin from HIT-T15 cells

We examined the effect of oxidized low-density lipoprotein (oxLDL) on the insulin secretion in the culture of HIT-T15 cell line, an islet beta-cell line derived from a hamster pancreatic tumor. In order to check the uptake of modified LDL by HIT-T15 cells, we prepared DiI-labeled native LDL (nLDL), acetylated LDL (AcLDL), and oxLDL. After the addition of each LDL into the cultures of HIT-T15 cells, fluorescence microscopic study was done. It was suggested that AcLDL and oxLDL were taken up by HIT-T15 cells, as well as nLDL. mRNA expression of the LDL receptor, CD36, and SR-B1 was detected in HIT-T15 by RT-PCR. The medium insulin level was measured in the culture of HIT-T15 cells with each LDL. oxLDL significantly reduced the insulin secretion stimulated by various concentrations of glucose, the intracellular content of insulin, and the expression of preproinsulin mRNA compared to the control cultures without LDL addition. In contrast, nLDL and AcLDL had no effect on the insulin secretion, the intracellular insulin level, or the expression of preproinsulin mRNA. MTT assay findings (reflecting cell numbers) were not different between cultures with and without LDLs. These results indicated that oxLDL disturbed the insulin metabolism of HIT-T15 cells.     

Increase in X-ray-induced mutations by exposure to magnetic field (60 Hz, 5 mT) in NF-kappaB-inhibited cells

It is established that extremely low frequency magnetic fields (ELFMF) at the flux densities, i.e., 5 mT and less, are not mutagenic. However, exposure to ELFMF enhances mutations induced by X-rays. In this study, we examined the effects of long-term exposure to 5 mT ELFMF on mutation induction and X-ray-induced mutations in human malignant glioma cells (MO54) with different mutant IkappaB-alpha (a critical inhibitor of NF-kappaB) genes. Cells were exposed or sham-exposed to 5 mT ELFMF for up to 8 days with or without initial X-rays (4 Gy), and the mutant frequency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was analyzed. An obvious increase in X-ray-induced mutations was observed after treatment with ELFMF in combination with X-irradiation in MO54 cells with tyrosine mutant IkappaB-alpha gene other than with serine mutant IkappaB-alpha gene or vector alone. Exposure to ELFMF alone increased mutations significantly in MO54 cells with tyrosine mutant IkappaB-alpha gene. In addition, X-ray-induced apoptoic cells were increased in MO54-V cells after exposure to ELFMF, while an anti-apoptotic effect of magnetic field was found in MO54-SY4 cells. Our data suggest that exposure to 5 mT ELFMF may induce mutations and enhance X-ray-induced mutations, resulting from the inactivation of NF-kappaB through the inhibition of tyrosine phosphorylation.     

Functional examination of microencapsulated bioengineered insulin-secreting beta-cells.

Clonal insulin-secreting BRIN-BD11 cells engineered by electrofusion were encapsulated inside natrium alginate beads and cultured in RPMI 1640 culture media. Acute insulin secretory responses to glucose and amino acids were compared between microencapsulated cells and non-encapsulated cells maintained in monolayer culture. Encapsulated cells exhibited a 1.5-fold, 2.9-fold and 4.2-fold increase (P< 0.001) in insulin release in response to 16.7 mmol/l glucose, 10 mmol/l L-arginine and 10 mmol/l L-alanine respectively. Insulin output by non-encapsulated cells was approximately 30% greater but the relative magnitudes of responses were similar. This is the first study to demonstrate the stability of cellular engineered insulin-secreting cells encapsulated in alginate beads, illustrating the utility of this approach for cellular engineering and potential transplantation in diabetes.     

Transplantation of insulin-secreting cells differentiated from human adipose tissue-derived stem cells into type 2 diabetes mice

Currently, there are limited ways to preserve or recover insulin secretory capacity in human pancreas. We evaluated the efficacy of cell therapy using insulin-secreting cells differentiated from human eyelid adipose tissue-derived stem cells (hEAs) into type 2 diabetes mice. After differentiating hEAs into insulin-secreting cells (hEA-ISCs) in vitro, cells were transplanted into a type 2 diabetes mouse model. Serum levels of glucose, insulin and c-peptide were measured, and changes of metabolism and inflammation were assessed in mice that received undifferentiated hEAs (UDC group), differentiated hEA-ISCs (DC group), or sham operation (sham group). Human gene expression and immunohistochemical analysis were done. DC group mice showed improved glucose level, and survival up to 60 days compared to those of UDC and sham group. Significantly increased levels of human insulin and c-peptide were detected in sera of DC mice. RT-PCR and immunohistochemical analysis showed human gene expression and the presence of human cells in kidneys of DC mice. When compared to sham mice, DC mice exhibited lower levels of IL-6, triglyceride and free fatty acids as the control mice. Transplantation of hEA-ISCs lowered blood glucose level in type 2 diabetes mice by increasing circulating insulin level, and ameliorating metabolic parameters including IL-6.     

Overexpression of Kruppel-like factor 7 regulates adipocytokine gene expressions in human adipocytes and inhibits glucose-induced insulin secretion in pancreatic beta-cell line

We have identified Kruppel-like factor 7 (KLF7) as a new candidate for conferring susceptibility to type 2 diabetes. To ascertain the possible involvement of KLF7 in the pathogenesis of type 2 diabetes, we examined the functional roles of KLF7 in various types of cells. In human adipocytes overexpressing KLF7, the expression of adiponectin and leptin was decreased compared with that in control cells, whereas expression of IL-6 was increased. In the insulin-secreting cell line (HIT-T15 cells), the expression and glucose-induced secretion of insulin were significantly suppressed in KLF7-overexpressed cells compared with control cells, accompanied by the reduction in the expression of glucose transporter 2, sulfonylurea receptor 1, Kir6.2, and pancreatic-duodenal homeobox factor 1. We also found that the overexpression of KLF7 resulted in the decrease of hexokinase 2 expression in smooth muscle cells, and of glucose transporter 2 expression in the HepG2 cells. These results suggest that KLF7 may contribute to the pathogenesis of type 2 diabetes through an impairment of insulin biosynthesis and secretion in pancreatic beta-cells and a reduction of insulin sensitivity in peripheral tissues. Therefore, we suggest that KLF7 plays an important role in the pathogenesis of type 2 diabetes, and may be a useful target for new drugs to aid in the prevention and treatment of this disease.     

Glucose increases cytosolic calcium concentration and inositol lipid metabolism in HIT-T15 cells

We have investigated the effects of glucose on cytosolic free calcium concentration in the insulin-secreting cell line HIT-T15. Addition of glucose (10 mM) caused a 20-75% increase in cytosolic [Ca2+] within 5 minutes compared to controls in the absence of glucose. A maximal increase in cytosolic [Ca2+] was obtained with 5 mM glucose. The magnitude of the response was markedly dependent upon the concentration of extracellular Ca2+, and the rise in cytosolic [Ca2+] was inhibited by verapamil. Cytosolic [Ca2+] was greatly increased by depolarization of the cells with KCl (50 mM), whereas carbamylcholine had no apparent effect. Glucose and KCl were also effective in stimulating insulin release from HIT cells, although carbamylcholine was again ineffective. The secretory response to glucose was also found to be directly related to the concentration of extracellular [Ca2+]. Glucose and KCl, but not carbamylcholine, were found to slightly enhance the production of [3H]-inositol trisphosphate in HIT cells pre-labelled with myo-[3H]-inositol, indicating a modest stimulation of inositol lipid hydrolysis.     

α-lipoic acid inhibits high glucose-induced apoptosis in HIT-T15 cells

High blood glucose plays an important role in the pathogenesis of diabetes. α-lipoic acid (LA) has been used to prevent and treat diabetes, and is thought to act by increasing insulin sensitivity in many tissues. However, whether LA also has a cytoprotective effect on pancreatic islet beta cells remains unclear. In this study, we assessed whether LA could inhibit apoptosis in beta cells exposed to high glucose concentrations. HIT-T15 pancreatic beta cells were treated with 30 mmol/L glucose in the presence or absence of 0.5 mmol/L LA for 8 days. LA significantly reduced the numbers of apoptotic HIT-T15 cells and inhibited the cell overgrowth normally induced by high glucose treatment. Additionally, LA inhibited insulin expression and secretion in HIT-T15 cells induced by high glucose. Further study demonstrated that LA upregulated Pdx1 and Bcl2 gene expression, reduced Bax gene expression, and promoted phosphorylation of Akt in HIT-T15 cells treated with high glucose. Intriguingly, knockdown of Pdx1 expression partially offset the anti-apoptotic effect of LA. However, inhibition of Akt by PI3K/AKT antagonist LY294002 only slightly reversed the anti-apoptosis effect of LA and mildly decreased the gene expression level of Pdx1 (P > 0.05). Moreover, LA only slightly attenuated reactive oxygen species (ROS) production and augmented mitochondrial membrane potential. Therefore, our data suggest that α-lipoic acid can effectively attenuate high glucose-induced HIT-T15 cell apoptosis probably by increasing Pdx1 expression. These findings provide a new interpretation on the role of LA in the treatment of diabetes.     

Determinants of glucose toxicity and its reversibility in the pancreatic islet beta-cell line, HIT-T15

HIT-T15 cells, a clonal beta-cell line, were cultured and passaged weekly for 6 mo in RPMI 1640 media containing various concentrations of glucose. Insulin content decreased in the intermediate- and late-passage cells as a continuous rather than a threshold glucose concentration effect. In a second series of experiments, cells were grown in media containing either 0.8 or 16.0 mM glucose from passages 76 through 105. Subcultures of passages 86, 92, and 99 that had been grown in media containing 16.0 mM glucose were switched to media containing 0.8 mM glucose and also carried forward to passage 105. Dramatic increases in insulin content and secretion and insulin gene expression were observed when the switches were made at passages 86 and 92 but not when the switch was made at passage 99. These findings suggest that glucose toxicity of insulin-secreting cells is a continuous rather than a threshold function of glucose concentration and that the shorter the period of antecedent glucose toxicity, the more likely that full recovery of cell function will occur.     

The dependence on intracellular ATP concentration of ATP-sensitive K-channels and of Na,K-ATPase in intact HIT-T15 beta-cells

We have studied the effects of changes of intracellular ATP concentration ([ATP]i) on the activity of ATP-sensitive K-channels (IK(ATP] and of Na,K-ATPase in intact cells of the insulin-secreting cell-line HIT-T15. Pre-exposure of HIT beta-cells to oligomycin caused a dose-dependent reduction in [ATP]i. Marked activation of IK(ATP) activity was found when ATP was lowered below 3 mM. Na,K-ATPase was progressively inhibited as ATP was lowered to 1.5 mM. These data demonstrate that changes in intracellular ATP in the millimolar range markedly influence the activity of two beta-cell membrane proteins having affinities for ATP in the micromolar range. This suggests that submembrane [ATP] may be considerably below the measured bulk cytosolic concentration. The findings also support the proposed role of intracellular ATP in mediating effects of changes in glucose concentration on the activity of beta-cell IK(ATP) and insulin secretion.     

Development of an optimized 5-stage protocol for the in vitro preparation of insulin-secreting cells from mouse ES cells

In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage. GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 10⁶ cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.     

Development of a bioartificial pancreas: II. Effects of oxygen on long-term entrapped betaTC3 cell cultures

Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes. An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation. Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct. In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium. Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state. Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR). Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)). Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR). A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels. It is concluded that (31)P NMR spectroscopy can be used for the prolonged noninvasive monitoring of the bioenergetic changes of encapsulated betaTC3 cells occurring with changes in oxygen tension. The data also indicate that the oxygen-dependent states might be related to the total number of viable, metabolically active cells supported by the particular oxygen level to which the system is exposed. These findings have significant implications in developing and non-invasively monitoring a tissue-engineered bioartificial pancreas based on transformed beta cells, as well as in understanding the biochemical events pertaining to insulin secretion from betaTC3 insulinomas.     

Generation of insulin-secreting cells from pancreatic acinar cells of animal models of type 1 diabetes

We recently found that pancreatic acinar cells isolated from normal adult mouse can transdifferentiate into insulin-secreting cells in vitro. Using two different animal models of type 1 diabetes, we show here that insulin-secreting cells can also be generated from pancreatic acinar cells of rodents in the diabetic state with absolute insulin deficiency. When pancreatic acinar cells of streptozotocin-treated mice were cultured in suspension in the presence of epidermal growth factor and nicotinamide under low-serum condition, expressions of insulin genes gradually increased. In addition, expressions of other pancreatic hormones, including glucagon, somatostatin, and pancreatic polypeptide, were also induced. Analysis by the Cre/loxP-based direct cell lineage tracing system revealed that these newly made cells originated from amylase-expressing pancreatic acinar cells. Insulin secretion from the newly made cells was significantly stimulated by high glucose and other secretagogues. In addition, insulin-secreting cells were generated from pancreatic acinar cells of Komeda diabetes-prone rats, another animal model of type 1 diabetes. The present study demonstrates that insulin-secreting cells can be generated by transdifferentiation from pancreatic acinar cells of rodents in the diabetic state and further suggests that pancreatic acinar cells represent a potential source of autologous transplantable insulin-secreting cells for treatment of type 1 diabetes.