Impact of treatment with saxagliptin on glycaemic stability and ß-cell function in the SAVOR-TIMI 53 study.

AIMS: To study the effects of saxagliptin, a dipeptidyl peptidase-4 inhibitor, on glycaemic stability and ß-cell function in the SAVOR-TIMI 53 trial. METHODS: We randomized 16,492 patients with type 2 diabetes (T2D) to saxagliptin or placebo, added to current antidiabetic medications, and followed them for a median of 2.1 years. Glycaemic instability was defined by: (i) a glycated haemoglobin (HbA1c) increase of ≥ 0.5% post-randomization; (ii) the initiation of new antidiabetic medications for ≥ 3 months; or (iii) an increase in dose of oral antidiabetic medication or ≥ 25% increase in insulin dose for ≥ 3 months. ß-cell function was assessed according to fasting homeostatic model 2 assessment of ß-cell function (HOMA-2ß) values at baseline and at year 2 in patients not treated with insulin. RESULTS: Compared with placebo, participants treated with saxagliptin had a reduction in the development of glycaemic instability (hazard ratio 0.71; 95% confidence interval 0.68-0.74; p < 0.0001). In participants treated with saxagliptin compared with placebo, the occurrence of an HbA1c increase of ≥ 0.5% was reduced by 35.2%; initiation of insulin was decreased by 31.7% and the increases in doses of an oral antidiabetic drug or insulin were reduced by 19.5 and 23.5%, respectively (all p < 0.0001). At 2 years, HOMA-2ß values decreased by 4.9% in participants treated with placebo, compared with an increase of 1.1% in those treated with saxagliptin (p < 0.0001). CONCLUSIONS: Saxagliptin improved glycaemia and prevented the reduction in HOMA-2ß values. Saxagliptin may reduce the usual decline in ß-cell function in T2D, thereby slowing diabetes progression.

Glucose regulation of ß-cell stress in type 2 diabetes.

In type 2 diabetes, the ß-cell is exposed to chronic hyperglycaemia, which increases its metabolic activity, with excess generation of reactive oxygen species (ROS) as a consequence. ROS accumulation induces both oxidative and endoplasmic reticulum (ER) stress, which may lead to ß-cell dysfunction and apoptosis. Recent data suggest that oxidative and ER stress are interconnected, although the mechanisms involved in nutrient regulation of the different stress pathways are dissimilar. Several components of the oxidative and ER stress machineries have important roles in the physiological response to glucose and are thus necessary for normal ß-cell function. Glucose stimulates signalling pathways that provide crucial messages for ß-cell adaptation to metabolic stress; however, the same pathways may eventually lead to apoptosis. Dynamic, temporally fluctuating activation of stress signalling is probably required for the maintenance of ß-cell survival, whereas its persistent activation results in ß-cell dysfunction and apoptosis. Thus, stress signalling is a 'double-edged sword' that may promote adaptation or apoptosis according to the balance between the divergent outputs of the various pathways. Developing new strategies for ß-cell protection based on inhibition of oxidative and/or ER stress requires comprehensive understanding of the switch from ß-cell adaptation to ß-cell apoptosis under conditions of metabolic stress, such as occurs under hyperglycaemic conditions.

Neuronal nitric oxide synthase protects the pancreatic beta cell from glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis.

AIMS/HYPOTHESIS: Cytokines stimulate nitric oxide production in pancreatic beta cells, leading to endoplasmic reticulum (ER) stress and apoptosis. Treatment of beta cells with glucose and NEFA induces nitric oxide synthase (NOS) as well as ER stress. However, the role of NO in glucolipotoxicity-induced ER stress in beta cells is not clear. METHODS: We studied the effect of high glucose and palmitate levels on NOS isoform production in rat and Psammomys obesus islets and in insulinoma-1E beta cells. The effects of neuronal NOS (nNOS) inhibition by small interfering RNA or by N (omega)-nitro-L-arginine methyl ester (L-NAME) on beta cell function, ER stress and apoptosis under conditions of glucolipotoxicity were investigated. RESULTS: Overnight incubation of rat and P. obesus islets at 22.2 mmol/l glucose with 0.5 mmol/l palmitate induced the production of nNOS but not inducible NOS (iNOS), in contrast with the robust stimulation of iNOS by cytokines. NOS inhibition by L-NAME did not prevent the decrease in glucose-stimulated insulin secretion and proinsulin biosynthesis or the depletion of islet insulin content observed under conditions of glucolipotoxicity. Moreover, treatment of beta cells with palmitate and L-NAME together resulted in marked activation of the IRE1alpha and PERK pathways of the unfolded protein response. This was associated with increased JNK phosphorylation and apoptosis in islets and beta cells. Moreover, partial nNos knockdown increased JNK phosphorylation and CHOP production, leading to apoptosis. CONCLUSIONS/INTERPRETATION: In beta cells subjected to glucolipotoxic conditions, chronic inhibition of NOS exacerbates ER stress and activates JNK. Therefore, induction of nNOS is an adaptive response to glucolipotoxicity that protects beta cells from stress and apoptosis.

Balancing needs and means: the dilemma of the beta-cell in the modern world.

The insulin resistance of type 2 diabetes mellitus (T2DM), although important for its pathophysiology, is not sufficient to establish the disease unless major deficiency of beta-cell function coexists. This is demonstrated by the fact that near-physiological administration of insulin (CSII) achieved excellent blood glucose control with doses similar to those used in insulin-deficient type 1 diabetics. The normal beta-cell adapts well to the demands of insulin resistance. Also in hyperglycaemic states some degree of adaptation does exist and helps limit the severity of disease. We demonstrate here that the mammalian target of rapamycin (mTOR) system might play an important role in this adaptation, because blocking mTORC1 (complex 1) by rapamycin in the nutritional diabetes model Psammomys obesus caused severe impairment of beta-cell function, increased beta-cell apoptosis and progression of diabetes. On the other hand, under exposure to high glucose and FFA (gluco-lipotoxicity), blocking mTORC1 in vitro reduced endoplasmic reticulum (ER) stress and beta-cell death. Thus, according to the conditions of stress, mTOR may have beneficial or deleterious effects on the beta-cell. beta-Cell function in man can be reduced without T2DM/impaired glucose tolerance (IGT). Prospective studies have shown subjects with reduced insulin response to present, several decades later, an increased incidence of IGT/T2DM. From these and other studies we conclude that T2DM develops on the grounds of beta-cells whose adaptation capacity to increased nutrient intake and/or insulin resistance is in the lower end of the normal variation. Inborn and acquired factors that limit beta-cell function are diabetogenic only in a nutritional/metabolic environment that requires high functional capabilities from the beta-cell.

Insulin counteracts glucotoxic effects by suppressing thioredoxin-interacting protein production in INS-1E beta cells and in Psammomys obesus pancreatic islets.

AIMS/HYPOTHESIS: In type 2 diabetes, glucose toxicity leads to beta cell apoptosis with decreased beta cell mass as a consequence. Thioredoxin-interacting protein (TXNIP) is a critical mediator of glucose-induced beta cell apoptosis. Since hyperglycaemia leads to elevated serum insulin, we hypothesised that insulin is involved in the regulation of TXNIP protein levels in beta cells. METHODS: We studied the production of TXNIP in INS-1E beta cells and in islets of Psammomys obesus, an animal model of type 2 diabetes, in response to glucose and different modulators of insulin secretion. RESULTS: TXNIP production was markedly augmented in islets from diabetic P. obesus and in beta cells exposed to high glucose concentration. In contrast, adding insulin to the culture medium or stimulating insulin secretion with different secretagogues suppressed TXNIP. Inhibition of glucose and fatty acid-stimulated insulin secretion with diazoxide increased TXNIP production in beta cells. Nitric oxide (NO), a repressor of TXNIP, enhanced insulin signal transduction, whereas inhibition of NO synthase abolished its activation, suggesting that TXNIP inhibition by NO is mediated by stimulation of insulin signalling. Treatment of beta cells chronically exposed to high glucose with insulin reduced beta cell apoptosis. Txnip knockdown mimicking the effect of insulin prevented glucose-induced beta cell apoptosis. CONCLUSIONS/INTERPRETATION: Insulin is a potent repressor of TXNIP, operating a negative feedback loop that restrains the stimulation of TXNIP by chronic hyperglycaemia. Repression of TXNIP by insulin is probably an important compensatory mechanism protecting beta cells from oxidative damage and apoptosis in type 2 diabetes.

The role of mTOR in the adaptation and failure of beta-cells in type 2 diabetes.

Mammalian target of rapamycin (mTOR) is an important nutrient sensor that plays a critical role in cellular metabolism, growth, proliferation and apoptosis and in the cellular response to oxidative stress. In addition, mTOR-raptor complex, also called mammalian target of rapamycin complex 1 (mTORC1), generates an inhibitory feedback loop on insulin receptor substrate proteins. It was suggested that nutrient overload leads to insulin/insulin-like growth factor 1 resistance in peripheral insulin-responsive tissues and in the beta-cells through sustained activation of mTORC1. In this review, we summarize the literature on the regulation and function of mTOR, its role in the organism's response to nutrients and its potential impact on lifespan, insulin resistance and the metabolic adaptation to hyperglycaemia in type 2 diabetes. We also propose a hypothesis based on data in the literature as well as data generated in our laboratory, which assigns a central positive role to mTOR in the maintenance of beta-cell function and mass in the diabetic environment.

Mitochondrial regulation of insulin production in rat pancreatic islets.

AIMS/HYPOTHESIS: The study was designed to identify the key metabolic signals of glucose-stimulated proinsulin gene transcription and translation, focusing on the mechanism of succinate stimulation of insulin production. METHODS: Wistar rat islets were incubated in 3.3 mmol/l glucose with and without esters of different mitochondrial metabolites or with 16.7 mmol/l glucose. Proinsulin biosynthesis was analysed by tritiated leucine incorporation into newly synthesised proinsulin. Preproinsulin gene transcription was evaluated following transduction with adenoviral vectors expressing the luciferase reporter gene under the control of the rat I preproinsulin promoter. Steady-state preproinsulin mRNA was determined using relative quantitative PCR. The mitochondrial membrane potential was measured by microspectrofluorimetry using rhodamine-123. RESULTS: Succinic acid monomethyl ester, but not other mitochondrial metabolites, stimulated preproinsulin gene transcription and translation. Similarly to glucose, succinate increased specific preproinsulin gene transcription and biosynthesis. The inhibitor of succinate dehydrogenase (SDH), 3-nitropropionate, abolished glucose- and succinate-stimulated mitochondrial membrane hyperpolarisation and proinsulin biosynthesis, indicating that stimulation of proinsulin translation depends on SDH activity. Partial inhibition of SDH activity by exposure to fumaric acid monomethyl ester abolished the stimulation of preproinsulin gene transcription, but only partially inhibited the stimulation of proinsulin biosynthesis by glucose and succinate, suggesting that SDH activity is particularly important for the transcriptional response to glucose. CONCLUSIONS/INTERPRETATION: Succinate is a key metabolic mediator of glucose-stimulated preproinsulin gene transcription and translation. Moreover, succinate stimulation of insulin production depends on its metabolism via SDH. The differential effect of fumarate on preproinsulin gene transcription and translation suggests that these processes have different sensitivities to metabolic signals.

Improvement of metabolic state in an animal model of nutrition-dependent type 2 diabetes following treatment with S 23521, a new glucagon-like peptide 1 (GLP-1) analogue.

Glucagon-like peptide 1 (GLP-1) analogues are considered potential drugs for type 2 diabetes. We studied the effect of a novel GLP-1 analogue, S 23521 ([a8-des R36] GLP-1-[7-37]-NH2), on the metabolic state and beta-cell function, proliferation and survival in the Psammomys obesus model of diet-induced type 2 diabetes. Animals with marked hyperglycaemia after 6 days of high-energy diet were given twice-daily s.c. injection of 100 microg/kg S 23521 for 15 days. Food intake was significantly decreased in S 23251-treated P. obesus; however, there was no significant difference in body weight from controls. Progressive worsening of hyperglycaemia was noted in controls, as opposed to maintenance of pre-treatment glucose levels in the S 23521 group. Prevention of diabetes progression was associated with reduced mortality. In addition, the treated group had higher serum insulin, insulinogenic index and leptin, whereas plasma triglyceride and non-esterified fatty acid levels were decreased. S 23521 had pronounced effect on pancreatic insulin, which was 5-fold higher than the markedly depleted insulin reserve of control animals. Immunohistochemical analysis showed islet degranulation with disrupted morphology in untreated animals, whereas islets from S 23521-treated animals appeared intact and filled with insulin; beta-cell apoptosis was approximately 70% reduced, without a change in beta-cell proliferation. S 23521 treatment resulted in a 2-fold increase in relative beta-cell volume. Overall, S 23521 prevented the progression of diabetes in P. obesus with marked improvement of the metabolic profile, including increased pancreatic insulin reserve, beta-cell viability and mass. These effects are probably due to actions of S 23521 both directly on islets and via reduced food intake, and emphasize the feasibility of preventing blood glucose deterioration over time in type 2 diabetes.

IPF1/PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal With type 2 diabetes.

The homeodomain transcription factor IPF1/PDX1 is required in beta-cells for efficient expression of insulin, glucose transporter 2, and prohormone convertases 1/3 and 2. Psammomys obesus, a model of diet-responsive type 2 diabetes, shows markedly depleted insulin stores when given a high-energy (HE) diet. Despite hyperglycemia, insulin mRNA levels initially remained unchanged and then decreased gradually to 15% of the basal level by 3 weeks. Moreover, insulin gene expression was not increased when isolated P. obesus islets were exposed to elevated glucose concentrations. Consistent with these observations, no functional Ipf1/Pdx1 gene product was detected in islets of newborn or adult P. obesus using immunostaining, Western blot, DNA binding, and reverse transcriptase-polymerase chain reaction analyses. Other beta-cell transcription factors (e.g., ISL-1, Nkx2.2, and Nkx6.1) were expressed in P. obesus islets, and the DNA binding activity of the insulin transcription factors RIPE3b1-Act and IEF1 was intact. Ipf1/Pdx1 gene transfer to isolated P. obesus islets normalized the defect in glucose-stimulated insulin gene expression and prevented the rapid depletion of insulin content after exposure to high glucose. Taken together, these results suggest that the inability of P. obesus islets to adapt to dietary overload, with depletion of insulin content as a consequence, results from IPF1/PDX1 deficiency. However, because not all animals become hyperglycemic on HE diet, additional factors may be important for the development of diabetes in this animal model.

beta-cell glucotoxicity in the Psammomys obesus model of type 2 diabetes.

Deficient insulin secretion and relative hyperproinsulinemia are characteristic features of type 2 diabetes. The gerbil Psammomys obesus appears to be an ideal natural model of the human disease because it shows increased tendency to develop diet-induced diabetes, which is associated with moderate obesity. The disease is characterized by initial hyperinsulinemia, progressing to hypoinsulinemia associated with depleted pancreatic insulin stores and an increased proportion of insulin precursor molecules in the blood and islets. Although the proinsulin translational efficacy was found to be increased in hyperglycemic animals, insulin mRNA levels were not augmented and exhibited a gradual decrease with disease progression. The development of hyperglycemia was associated with a transient increase in beta-cell proliferative activity, as opposed to a prolonged increase in the rate of beta-cell death, culminating in disruption of islet architecture. The hypothesis that glucotoxicity is responsible in part for these in vivo changes was investigated in vitro in primary islet cultures. Islets from diabetes-prone P. obesus cultured at high glucose concentrations displayed changes in beta-cell function that mimic those observed in diabetic animals. These changes include deficient insulin secretion, depleted insulin content, an increased proportion of insulin precursor molecules, a progressive increase of DNA fragmentation, and a transient proliferative response. Furthermore, insulin mRNA was not increased by short-term exposure of P. obesus islets to elevated glucose in vitro. It is proposed that beta-cell glucotoxicity in P. obesus results from the inability of proinsulin biosynthesis to keep pace with chronic insulin hypersecretion. The resulting depletion of the insulin stores may be related to deficient glucose-regulated insulin gene transcription, possibly due to defective PDX-1 (pancreatic duodenal homeobox factor-1) expression in the adult P. obesus. An additional glucotoxic effect involves the loss of beta-cell mass in hyperglycemic P. obesus as a result of progressive beta-cell death without an adequate increase in the rate of beta-cell proliferation.

Protection from cell death in cultured human fetal pancreatic cells.

Endocrine cells from the human fetal pancreas will proliferate in vitro on extracellular matrix but lose hormone expression, and redifferentiation requires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation and culture. The mechanism of cell death was examined at each stage throughout the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of trehalose to the media to prevent necrosis increased yield 17-fold, while during culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increased yield 1.8-fold. Following disruption of cell matrix interactions and reaggregation, there was marked evidence of apoptotic bodies by the TUNEL assay. Addition of nicotinamide or Z-VAD, or removal of arginine from the media during reaggregation, reduced the number of apoptotic bodies and the effect was additive. However, a combination of treatments was necessary to significantly increase the yield of viable cells. We conclude that cell death of human fetal pancreatic tissue in culture results from both necrosis and apoptosis and that understanding the mechanisms at the cellular level will lead to protocols that will improve cell viability and promote beta-cell growth.

[Type 2 diabetes and beta cell apoptosis]. / Diabéte de type 2 et apoptose des cellules beta.

Type 2 diabetes mellitus features an asymptomatic insulin resistance phase preceding the onset of diabetes. Hyperglycemia occurs when a relative insulin deficiency appears, meaning that beta cell secretory dysfunction is a key element in type 2 diabetes pathophysiology. So far, insulin secretion deficiency is explained by pancreatic beta cell "exhaustion" phenomena. Recent data suggest that apoptotic mechanisms could explain insulin deficiency through a reduction in the absolute pancreatic beta cell number. Psammomys obesus (sand rat) is an animal model for type 2 diabetes mellitus, initially characterized by hyperinsulinism followed by insulin deficiency linked with a reduction in the number of pancreatic beta cells. Transition to diabetes can be observed following changes in usual lifestyle of the sand rat. In the desert, caloric intake is low and physical expenditure is heavy. In the laboratory, animals turn diabetic as early as 4 days following a high calorie diet. At a later stage, diabetes is irreversible and animals die from diabetic ketoacidosis. beta cell apoptosis rate is low in non diabetic animals and increases 14-fold by 20 days after diabetes onset. At this stage, cells undergoing apoptosis can be observed, coexisting with necrotic cells without any insulitis. Similar results were obtained in vitro in isolated pancreatic islets that were exposed to increasing glucose concentrations, suggesting that chronic hyperglycemia plays a role in the onset or the deterioration of the process. However, precise mechanisms of apoptosis in this case remain poorly understood. Aminoguanidin does not prevent beta cell apoptosis in vitro, suggesting that advanced glycation products or NO production are not involved in this beta cell destruction process. Similar mechanisms secondary to hyperglycemia could play a role in the diabetes process in man and explain the marked insulin secretory deficiency that is sometimes observed in these patients. In addition to its preventing role on diabetes complication, the obtention of normoglycemia could help maintaining beta cell function.

Sustained proliferation of PDX-1+ cells derived from human islets.

Ex vivo expansion of human beta-cells is an important step toward the development of cell-based insulin delivery systems in type 1 diabetes. Here, we report that human pancreatic endocrine cells can be expanded through 15 cell doublings in vitro for an estimated total 30,000-fold increase in cell number. We believe that the cells resulting from these cultures are of beta-cell origin, since they uniformly express the transcription factor PDX-1 (STF-1, IDX-1, IPF-1), which is initially seen only in cells positive for insulin and negative for the ductal cell marker cytokeratin (CK)-19. To rule out the possibility that PDX-1 expression might be induced by the culture conditions used here, cells from isolated human pancreatic ducts were cultured under the same conditions as the islet cells. Cells in these cultures expressed CK-19 but not PDX-1. Although the expanded beta-cells continued to express PDX-1, insulin expression was lost over time. Whether reexpression of islet-specific genes in vitro is essential for successful cell transplantation remains to be determined.

Impaired beta-cell functions induced by chronic exposure of cultured human pancreatic islets to high glucose.

In type 2 diabetes, chronic hyperglycemia has been suggested to be detrimental to beta-cell function, causing reduced glucose-stimulated insulin secretion and disproportionately elevated proinsulin. In the present study, we investigated the effect on several beta-cell functions of prolonged in vitro exposure of human pancreatic islet cultures to high glucose concentrations. Islets exposed to high glucose levels (33 mmol/l) for 4 and 9 days showed dramatic decreases in glucose-induced insulin release and in islet insulin content, with increased proportion of proinsulin-like peptides relative to insulin. The depletion in insulin stores correlated with the reduction in insulin mRNA levels and human insulin promoter transcriptional activity. We also demonstrated that high glucose dramatically lowered the binding activity of pancreatic duodenal homeobox 1 (the glucose-sensitive transcription factor), whereas the transcription factor rat insulin promoter element 3b1 activator was less influenced and insulin enhancer factor 1 remained unaffected. Most of these beta-cell impairments were partially reversible when islets first incubated for 6 days in high glucose were transferred to normal glucose (5.5 mmol/l) concentrations for 3 days. We conclude that cultured human islets are sensitive to the deleterious effect of high glucose concentrations at multiple functional levels, and that such mechanisms may play an important role in the decreased insulin production and secretion of type 2 diabetic patients.

Presenilin 1 facilitates the constitutive turnover of beta-catenin: differential activity of Alzheimer's disease-linked PS1 mutants in the beta-catenin-signaling pathway.

Although an association between the product of the familial Alzheimer's disease (FAD) gene, presenilin 1 (PS1), and beta-catenin has been reported recently, the cellular consequences of this interaction are unknown. Here, we show that both the full length and the C-terminal fragment of wild-type or FAD mutant PS1 interact with beta-catenin from transfected cells and brains of transgenic mice, whereas E-cadherin and adenomatous polyposis coli (APC) are not detected in this complex. Inducible overexpression of PS1 led to increased association of beta-catenin with glycogen synthase kinase-3beta (GSK-3beta), a negative regulator of beta-catenin, and accelerated the turnover of endogenous beta-catenin. In support of this finding, the beta-catenin half-life was dramatically longer in fibroblasts deficient in PS1, and this phenotype was completely rescued by replacement of PS1, demonstrating that PS1 normally stimulates the degradation of beta-catenin. In contrast, overexpression of FAD-linked PS1 mutants (M146L and DeltaX9) failed to enhance the association between GSK-3beta and beta-catenin and interfered with the constitutive turnover of beta-catenin. In vivo confirmation was demonstrated in the brains of transgenic mice in which the expression of the M146L mutant PS1 was correlated with increased steady-state levels of endogenous beta-catenin. Thus, our results indicate that PS1 normally promotes the turnover of beta-catenin, whereas PS1 mutants partially interfere with this process, possibly by failing to recruit GSK-3beta into the PS1-beta-catenin complex. These findings raise the intriguing possibility that PS1-beta-catenin interactions and subsequent activities may be consequential for the pathogenesis of AD.

Towards gene therapy of diabetes mellitus.

A definitive treatment for diabetes mellitus will be one that maintains a normal blood glucose concentration in the face of fluctuating dietary intake. To accomplish this, there must be mechanisms to sense the amount of blood glucose coupled to rapid release of the right amount of insulin. While mechanical devices to accomplish this are being developed, ultimately the best approach is likely to be based on genetic modification of cells. These could be pancreatic beta-cells, which are the only cells that produce insulin, or other cells that are involved in the pathogenesis of diabetes. Although definitive treatment of diabetes using genetically modified cells is a long-term goal, much progress is being made. This review discusses various approaches to modifying cells genetically, both in vitro and in vivo, for the treatment of diabetes.

Gene transfer to human pancreatic endocrine cells using viral vectors.

We have studied the factors that influence the efficiency of infection of human fetal and adult pancreatic endocrine cells with adenovirus, murine retrovirus, and lentivirus vectors all expressing the green fluorescent protein (Ad-GFP, MLV-GFP, and Lenti-GFP, respectively). Adenoviral but not retroviral vectors efficiently infected intact pancreatic islets and fetal islet-like cell clusters (ICCs) in suspension. When islets and ICCs were plated in monolayer culture, infection efficiency with all three viral vectors increased. Ad-GFP infected 90-95% of the cells, whereas infection with MLV-GFP and Lenti-GFP increased only slightly. Both exposure to hepatocyte growth factor/scatter factor (HGF/SF) and dispersion of the cells by removal from the culture dish and replating had substantial positive effects on the efficiency of infection with retroviral vectors. Studies of virus entry and cell replication revealed that cell dispersion and stimulation by HGF/SF may be acting through both mechanisms to increase the efficiency of retrovirus-mediated gene transfer. Although HGF/SF and cell dispersion increased the efficiency of infection with MLV-GFP, only rare cells with weak staining for insulin were infected, whereas approximately 25% of beta-cells were infected with Lenti-GFP. We conclude that adenovirus is the most potent vector for ex vivo overexpression of foreign genes in adult endocrine pancreatic cells and is the best vector for applications where high-level but transient expression is desired. Under the optimal conditions of cell dispersion plus HGF/SF, infection with MLV and lentiviral vectors is reasonably efficient and stable, but only lentiviral vectors efficiently infect pancreatic beta-cells.

Telomerase activity is sufficient to allow transformed cells to escape from crisis.

The introduction of simian virus 40 large T antigen (SVLT) into human primary cells enables them to proliferate beyond their normal replicative life span. In most cases, this temporary escape from senescence eventually ends in a second proliferative block known as "crisis," during which the cells cease growing or die. Rare immortalization events in which cells escape crisis are frequently correlated with the presence of telomerase activity. We tested the hypothesis that telomerase activation is the critical step in the immortalization process by studying the effects of telomerase activity in two mortal SVLT-Rasval12-transformed human pancreatic cell lines, TRM-6 and betalox5. The telomerase catalytic subunit, hTRT, was introduced into late-passage cells via retroviral gene transfer. Telomerase activity was successfully induced in infected cells, as demonstrated by a telomerase repeat amplification protocol assay. In each of nine independent infections, telomerase-positive cells formed rapidly dividing cell lines while control cells entered crisis. Telomere lengths initially increased, but telomeres were then maintained at their new lengths for at least 20 population doublings. These results demonstrate that telomerase activity is sufficient to enable transformed cells to escape crisis and that telomere elongation in these cells occurs in a tightly regulated manner.