Karyotype of the human insulinoma CM cell line—Beta cell model in vitro?

The CM cell line is derived from a human pancreatic insulinoma and is used as a beta cell model for the study of the pathogenesis of diabetes, as it appears to maintain the characteristics of beta cells. However, a karyotype study of the CM cell line was not previously performed. We aimed at karyotyping the CM cell line to confirm its human origin, diploid karyotype, and chromosomal structure. We karyotyped the CM cells at earlier passages with the standard Giemsa technique. The karyotyping procedure confirmed the human origin of the CM cell line. However, the karyotype showed 64 chromosomes with structural abnormalities, including chromosome 11, in which the insulin gene is located. Our Medline search of other existing insulinoma cell lines of rodent, mouse and hamster origin did not show any karyotype performed. As the CM cell karyotype reveals significant structural and numerical chromosomal abnormalities, we question the use of such a cell line as an in vitro beta cell model. We suggest that insulinoma cell lines established in vitro to study beta cell function should have a karyotype performed to exclude chromosomal aberrations.     

Regulation of immunoreactive-insulin release from a rat cell line (RINm5F).

1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.     

Taurine regulates insulin release from pancreatic beta cell lines

Background

Pancreatic β-cells release insulin via an electrogenic response triggered by an increase in plasma glucose concentrations. The critical plasma glucose concentration has been determined to be ~3 mM, at which time both insulin and GABA are released from pancreatic β-cells. Taurine, a β-sulfonic acid, may be transported into cells to balance osmotic pressure. The taurine transporter (TauT) has been described in pancreatic tissue, but the function of taurine in insulin release has not been established. Uptake of taurine by pancreatic β-cells may alter membrane potential and have an effect on ion currents. If taurine uptake does alter β-cell current, it might have an effect on exocytosis of cytoplasmic vesicle. We wished to test the effect of taurine on regulating release of insulin from the pancreatic β-cell.

Methods

Pancreatic β-cell lines Hit-TI5 (Syrian hamster) and Rin-m (rat insulinoma) were used in these studies. Cells were grown to an 80% confluence on uncoated cover glass in RPMI media containing 10% fetal horse serum. The cells were then adapted to a serum-free, glucose free environment for 24 hours. At that time, the cells were treated with either 1 mM glucose, 1 mM taurine, 1 mM glucose + 1 mM taurine, 3 mM glucose, or 3 mM glucose + 1 mM taurine. The cells were examined by confocal microscopy for cytoplasmic levels of insulin.

Results

In both cell lines, 1 mM glucose had no effect on insulin levels and served as a control. Cells starved of glucose had a significant reduction (p<0.001) in the level of insulin, but this level was significantly higher than all other treatments. As expected, the 3 mM glucose treatment resulted in a statistically lower (p<0.001) insulin level than control cells. Interestingly, 1 mM taurine also resulted in a statistically lower level of insulin (p<0.001) compared to controls when either no glucose or 1 mM glucose was present. Cells treated with 1 mM taurine plus 3 mM glucose showed a level of insulin similar to that of 3 mM glucose alone.

Conclusions

Taurine administration can alter the electrogenic response in β-cell lines, leading to a change in calcium homeostasis and a subsequent decrease in intracellular insulin levels. The consequence of these actions could represent a method of increasing plasma insulin levels leading to a decrease in plasma glucose levels.

     

Transport and metabolism of glucose in an insulin-secreting cell line, beta TC-1.

Kinetic characteristics of glucose transport and glucose phosphorylation were studied in the islet cell line beta TC-1 to explore the roles of these processes in determining the dependence of glucose metabolism and insulin secretion on external glucose. The predominant glucose transporter present was the rat brain/erythrocyte type (Glut1), as determined by RNA and immunoblot analysis. The liver/islet glucose transporter (Glut2) RNA was not detected. The functional parameters of zero-trans glucose entry were Km = 9.5 +/- 2 mM and Vmax = 15.2 +/- 2 nmol min-1 (microL of cell water)-1. Phosphorylation kinetics of two hexokinase activities were characterized in situ. A low-Km (0.036 mM) hexokinase with a Vmax of 0.40 nmol min-1 (microL of cell water)-1 was present along with a high-Km (10 mM) hexokinase, which appeared to conform to a cooperative model with a Hill coefficient of about 1.4 and a Vmax of 0.3 nmol min-1 (microL of cell water)-1. Intracellular glucose at steady state was about 80% of the extracellular glucose from 3 to 15 mM, and transport did not limit metabolism in this range. In this static (nonperifusion) system, 2-3 times more immunoreactive insulin was secreted into the medium at 15 mM glucose than at 3 mM. The dependence of insulin secretion on external glucose roughly paralleled the dependence of glucose metabolism on external glucose. Simulations with a model demonstrated the degree to which changes in transport activity would affect intracellular glucose levels and the rate of the high-Km hexokinase (with the potential to affect insulin release).     

Regulation of insulin mRNA abundance and adenylation: dependence on hormones and matrix substrata.

The insulin mRNA levels of rat insulinoma cell lines increased six- to eightfold, and the cells entered a transient state of growth arrest when they were cultured in serum-free, hormonally defined medium and on an extract of extracellular matrix derived from a basement membrane-secreting tumor line, EHS. Insulinoma cultures in growth arrest responded to glucose with a two- to threefold increase in insulin secretion associated with an insulin mRNA that contained a poly(A) tail that was 120 to 140 bases longer than normal.     

Relative hypoglycemia and hyperinsulinemia in mice with heterozygous lipoprotein lipase (LPL) deficiency. Islet LPL regulates insulin secretion.

Lipoprotein lipase (LPL) provides tissues with fatty acids, which have complex effects on glucose utilization and insulin secretion. To determine if LPL has direct effects on glucose metabolism, we studied mice with heterozygous LPL deficiency (LPL+/-). LPL+/- mice had mean fasting glucose values that were up to 39 mg/dl lower than LPL+/+ littermates. Despite having lower glucose levels, LPL+/- mice had fasting insulin levels that were twice those of +/+ mice. Hyperinsulinemic clamp experiments showed no effect of genotype on basal or insulin-stimulated glucose utilization. LPL message was detected in mouse islets, INS-1 cells (a rat insulinoma cell line), and human islets. LPL enzyme activity was detected in the media from both mouse and human islets incubated in vitro. In mice, +/- islets expressed half the enzyme activity of +/+ islets. Islets isolated from +/+ mice secreted less insulin in vitro than +/- and -/- islets, suggesting that LPL suppresses insulin secretion. To test this notion directly, LPL enzyme activity was manipulated in INS-1 cells. INS-1 cells treated with an adeno-associated virus expressing human LPL had more LPL enzyme activity and secreted less insulin than adeno-associated virus-beta-galactosidase-treated cells. INS-1 cells transfected with an antisense LPL oligonucleotide had less LPL enzyme activity and secreted more insulin than cells transfected with a control oligonucleotide. These data suggest that islet LPL is a novel regulator of insulin secretion. They further suggest that genetically determined levels of LPL play a role in establishing glucose levels in mice.     

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.     

Insulin release from BK virus-induced and in vitro maintained insulinoma cells of Syrian golden hamster

The pancreatic tumor cells (In 111) derived from BK virus-induced insulinoma of Syrian golden hamsters were maintained in culture for several passages and were studied for their insulin secretory ability under various stimulatory conditions. Insulin release was not increased by D-glucose stimulation (27.8 mM), while dibutyryl cyclic AMP (1 mM), theophylline (1 mM), 3-isobutyl-l-methylxanthine (0.1 mM) and elevation of medium calcium from 0.5 to 2.7 mM stimulated insulin release 2.5- to 4-fold. There was a concomitant increase of medium cyclic AMP with addition of theophylline. Streptozotocin (2 mM) treatment for 48 hours significantly reduced insulin release, while alloxan (2 mM), had no inhibitory effect on insulin release. The results indicate that while in vitro-maintained islet tumor cells, In 111, have a cyclic AMP-mediated process involved in insulin secretion analogous to normal beta cells, these cells lack the ability to recognize glucose as an insulin secretagogue probably due to a defect in the cell membrane, though the possibility of alteration in glucose metabolism cannot be fully excluded.     

Morphologic and functional study of a human insulin-secreting cell line

Monolayer cell cultures were obtained from a human insulinoma (HIN) after collagenase digestion. HIN cells were initially plated on extracellular matrix (ECM) secreted by bovine corneal endothelial cells. Capsular integrity from cell clusters quickly interrupted and cell began to migrate as adhesive sheets onto ECM. After 2 months on ECM cell attachment and proliferation occurred on plastic allowing cloning of cells by limiting dilution. 9 clones were successfully cultured for 7 months with 20 subsequent passages. Immunoreactivity for insulin by indirect immunofluorescence typical secretory granules by electron microscopy and stable amounts of immunoreactive insulin in culture media suggest that HIN cells are beta cell related. One clone HIN D8 when challenged for half an hour with either 30 mM glucose, 1 mM isobutyl Methylxanthine 4 mM Tolbutamide, 10(-6) M glucagon responded respectively with a 1.5, 2, 3 and 1.5 fold increase in insulin output. Population doubling time of HIN D8 was 42 hrs. Establishment of such insulin secreting cell lines provides a valuable tool for diabetes research.     

Detecting de novo insulin synthesis in embryonic stem cell-derived populations

Several studies in recent years have described protocols, both genetic- and culture-based, that induce the differentiation of embryonic stem (ES) cells towards a pancreatic beta-cell type. The success of previous protocols in generating insulin-producing beta-cells has been questioned due in part to uncertainty regarding cell lineage but also due to the controversy regarding the source of any insulin detected in these cells. In an attempt to address the latter, we designed a novel assay that can identify de novo insulin synthesis. The method is based on metabolic labeling combined with a modified radio-immunoassay and will routinely detect less than 5 pg/microl of de novo insulin synthesis in lysates from the insulinoma cell line MIN6. This assay failed to detect any newly translated insulin in an ES cell-derived population generated using an adapted version of a previously published, 5-stage differentiation protocol. In combination with other techniques, including immunofluorescent staining and western blot analysis to detect and quantify C-peptide, we conclude that the majority of the insulin found in these differentiated ES cell cultures is medium-derived.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.     

Phenotypic characteristics of cell lines requiring interleukin 3 for growth

A number of cell lines have been derived from bone marrow cultures in the presence of WEHI-3 conditioned media (CM) that continue to require WEHI-3 CM for growth in vitro. Because the WEHI-3 cell line has been shown to constitutively produce a lymphokine (IL 3) that induces the expression of 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of splenic lymphocytes from athymic mice, we examined whether these cell lines were dependent upon IL 3 for growth. The results demonstrate that the factor required for growth of these cell lines copurifies with IL 3 activity on G-100, DEAE cellulose, and CM cellulose column chromatography as well as in preparative isoelectric focusing and on hydrophobic supports in reverse phase high pressure liquid chromatography. The biologic activity of peak fractions in each case was similar in both types of assays. These results strongly suggest that the WEHI-3 CM-dependent cell lines are dependent on IL 3 for growth in vitro. All the cell lines have readily detectable levels of 20 alpha SDH but have differing cell surface phenotypes. The C3HSFFV line is devoid of conventional lymphoid cell surface markers with the exception of Lyt-1, whereas the FDC-P1 expresses Thy-1, Ly-5, and H-11. Other cell lines have intermediate phenotypes.     

Glucose-6-phosphatase in the insulin secreting cell line INS-1

The glucose-6-phosphatase system of the glucose sensitive insulin secreting rat insulinoma cells (INS-1) was investigated. INS-1 cells contain easily detectable levels of glucose-6-phosphatase enzyme protein (assessed by Western blotting) and have a very significant enzymatic activity. The features of the enzyme (Km and Vmax values, sensitivity to acidic pH, partial latency, and double immunoreactive band) are similar to those of the hepatic form. On the other hand, hardly detectable levels of glucose-6-phosphatase activity and protein were present in the parent glucose insensitive RINm5F cell line. The mRNA of the glucose-6-phosphate transporter was also more abundant in the INS-1 cells. The results support the view that the glucose-6-phosphatase system of the beta-cell is associated with the regulation of insulin secretion.     

Biosynthesis of insulin secretory granule membrane proteins. Control by glucose.

The biosynthesis of a component SGM 110, specifically localized to the membrane of insulin secretory granules, was studied in rat insulinoma cells and in normal islets of Langerhans. Cells or islets were labelled with [35S]methionine or [3H]mannose and SGM 110 was immunoprecipitated by using a monoclonal antibody. Pulse-chase experiments demonstrated that the nascent polypeptide was cotranslationally glycosylated to form a 97,000 Da peptide which in turn was processed to the mature 110,000 Da form. A 50,000 Da form detected by immunoblotting with the same antibody was not conspicuously labelled even after a 20 h chase incubation, suggesting that it represented late processing of SGM 110 in lysosomes. With insulinoma cells, an increase in medium glucose concentration from 3 mM to 20 mM was without effect on the secretion of insulin or on the biosynthesis of (pro)insulin or SGM 110. In normal islets, however, 20 mM-glucose produced a 17-fold increase in (pro)insulin biosynthesis and a 13-fold increase in SGM 110 biosynthesis, compared with only a 2-fold increase in total protein synthesis, as judged by incorporation of [35S]methionine during a 1 h incubation. The effect of glucose on both (pro)insulin and SGM 110 biosynthesis was blocked by the addition of mannoheptulose, but not by the removal of extracellular calcium, both of which conditions inhibit insulin secretion. In contrast tolbutamide, an agent which stimulates insulin secretion, did not enhance the biosynthesis of (pro)insulin or SGM 110. It is concluded that at least one protein component of the insulin secretory granule membrane is synthesized co-ordinately with proinsulin and is subject to similar regulatory mechanisms. Factors which acutely control insulin secretion may also control granule biogenesis, although the two processes are not coupled in an obligatory fashion.     

Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.     

Insulin and islet cell transplantation in streptozotocin-diabetic rats: effect on intestinal uptake of hexoses

A previously validated in vitro technique was used to determine the effect of once daily injections of NPH insulin (NPH) and/or islet cell transplantation on the jejunal uptake of 0.5-40 mM glucose and galactose into the jejunum of streptozotocin-diabetic rats. Glucose uptake was greater in untreated diabetic rats than in control animals due to a higher maximal transport rate and a higher passive permeability of the jejunum, and a lower value of the apparent Michaelis constant. Galactose uptake was greater in diabetic rats due to a higher maximal transport rate, but there was also a higher value of the apparent Michaelis constant. This enhanced uptake of glucose and galactose was reduced and normalized by daily injections of NPH insulin or by islet cell transplantation. It is concluded: the jejunal uptake of glucose and galactose is increased in diabetic rats, but the kinetic basis for this change was different for the two sugars; insulin therapy may correct the enhanced uptake of some nutrients in diabetic rats and islet cell transplantation may be at least as effective as exogenous insulin in modifying the intestinal adaptation to diabetes.     

Homocysteine and other structurally-diverse amino thiols can alter pancreatic beta cell function without evoking cellular damage

Homocysteine and related amino thiols, homocysteic acid, cysteic acid, homocysteine sulphinic acid and cysteine sulphinic acid have been labelled as neurotoxins. Homocysteine thiolactone, a metabolic derivative of homocysteine, is cytotoxic to endothelial cells and other cell lineages. Since pancreatic beta cells share many phenotypic similarities with neuronal cells, the present study uses clonal pancreatic BRIN-BD11 cells to investigate possible detrimental effects of these amino thiols on insulin secretion and pancreatic beta cell function. Insulin secretion was concentration-dependently inhibited at both basal (1.1 mM) and stimulatory (16.7 mM) glucose by homocysteine, homocysteine thiolactone and homocysteine sulphinic acid. Cysteic acid concentration-dependently inhibited insulin secretion at 16.7 mM glucose. Cell viability was not compromised by any of the amino thiols. Insulin secretory responses to alanine were inhibited by homocysteine, homocysteine thiolactone, homocysteic acid and cysteic acid. Insulin secretion in the presence of elevated Ca(2+) and forskolin were lowered by all amino thiols, except homocysteic acid. The secretory responsiveness to PMA, GLP-1 and KCl were only impaired in the presence of homocysteine and homocysteine thiolactone. These findings indicate that homocysteine, homocysteine thiolactone and, to a lesser extent, other amino thiols cause dysfunctional insulin secretion from pancreatic beta cells.