Morphology and functional responses of isolated inner adrenocortical cells of rats infused with interleukin-beta.

The effects of the prolonged infusion with interleukin-1 beta (IL-1 beta) (20 pM.kg-1.min-1) on the function and morphology of the isolated inner cells of the rat adrenal cortex were investigated. After 3 and 5 days of IL-1 beta infusion, the level of circulating ACTH was below the control level, while the plasma concentration of corticosterone was strikingly elevated. After 5 days of infusion, isolated inner adrenocortical cells showed an enhanced basal and ACTH-stimulated corticosterone secretion, and showed a conspicuous hypertrophy. The acute exposure to IL-1 beta 10(-6) M did not affect the secretory activity of dispersed cell from either control or IL-1 beta-infused rats. These findings indicate that the prolonged exposure to high levels of circulating IL-1 beta, like those occurring during chronic inflammatory diseases, is able to enhance the growth and steroidogenic (glucocorticoid) capacity of the rat inner adrenocortical zones. Moreover, they suggest that the mechanism underlying this adrenocorticotrophic effect of IL-1 beta does not involve either a stimulation of the hypophyseal ACTH release or a direct stimulatory effect of monokine on adrenocortical cells. It is suggested that IL-1 beta may activate an intra-adrenal paracrine regulatory mechanism.     

Characterization of an Insulin Receptor in Human Y79 Retinoblastoma Cells

Cultured human Y79 retinoblastoma cells bind [125I]iodoinsulin in a manner similar to that of other CNS and peripheral tissues. The only difference noted between the insulin binding properties of the Y79 cells and other CNS preparations is that insulin binding to Y79 cells is down-regulated by prolonged exposure of the cells to insulin. By contrast, studies with the various brain preparations indicate that the brain insulin receptor is not down-regulated by circulating levels of insulin. Insulin binding to Y79 cells exhibits negative cooperativity, has a pH optimum of 7.8, is responsive to cations, and gives a curvilinear Scatchard plot. Y79 cell insulin binding capacity is 26 fmol/100 micrograms of cell protein, corresponding to about 125,000 binding sites per cell. These findings are the first to report insulin binding in a human cell line of retinal origin. The characterization of the insulin binding in this cell line may facilitate an understanding of the relationship between insulin and its specific functions in the human retina.     

High glucose and free fatty acids induce beta cell apoptosis via autocrine effects of ADP acting on the P2Y13 receptor

While high levels of glucose and saturated fatty acids are known to have detrimental effects on beta cell function and survival, the signalling pathways mediating these effects are not entirely known. In a previous study, we found that ADP regulates beta cell insulin secretion and beta cell apoptosis. Using MIN6c4 cells as a model system, we investigated if autocrine/paracrine mechanisms of ADP and purinergic receptors are involved in this process. High glucose (16.7 mmol/l) and palmitate (100 μmol/l) rapidly and potently elevated the extracellular ATP levels, while mannitol was without effect. Both tolbutamide and diazoxide were without effect, while the calcium channel blocker nifedipine, the volume-regulated anion channels (VRAC) inhibitor NPPB, and the pannexin inhibitor carbenoxolone could inhibit both effects. Similarly, silencing the MDR1 gene also blocked nutrient-generated ATP release. These results indicate that calcium channels and VRAC might be involved in the ATP release mechanism. Furthermore, high glucose and palmitate inhibited cAMP production, reduced cell proliferation in MIN6c4 and increased activated Caspase-3 cells in mouse islets and in MIN6c4 cells. The P2Y₁₃-specific antagonist MRS2211 antagonized all these effects. Further studies showed that blocking the P2Y₁₃ receptor resulted in enhanced CREB, Bad and IRS-1 phosphorylation, which are known to be involved in beta cell survival and insulin secretion. These findings provide further support for the concept that P2Y₁₃ plays an important role in beta cell apoptosis and suggest that autocrine/paracrine mechanisms, related to ADP and P2Y₁₃ receptors, contribute to glucolipotoxicity.     

Expression of adiponectin receptors in pancreatic beta cells

Pancreatic beta cell dysfunction is an early and crucial pathogenic factor in the development of type 2 diabetes. Free fatty acids (FFA) and adipokines released from adipose tissues lead to both the development of insulin resistance and beta cell dysfunction. Adiponectin is a novel adipokine with antidiabetic properties. Its circulating concentrations are reduced in subjects with increased visceral adiposity, insulin resistance, or type 2 diabetes. Very recently, the cloning of two adiponectin receptors AdipoR1 and AdipoR2 was reported. AdipoR1 is abundantly expressed in muscle, while AdipoR2 is predominantly expressed in liver. Here we report the marked expression of mRNAs for the adiponectin receptors AdipoR1 and AdipoR2 in human and rat pancreatic beta cells, at levels similar to liver and greater than muscle. Adiponectin receptor expression is increased by beta cell exposure to the unsaturated FFA oleate, and treatment of insulin-producing cells with globular adiponectin induces lipoprotein lipase expression. Regulated adiponectin receptor expression on pancreatic beta cells might be a novel mechanism modulating the effects of circulating adiponectin.     

Specificity in beta cell expression of L-3-hydroxyacyl-CoA dehydrogenase, short chain, and potential role in down-regulating insulin release

A loss-of-function mutation of the mitochondrial beta-oxidation enzyme l-3-hydroxyacyl-CoA dehydrogenase, short chain (HADHSC), has been associated with hyperinsulinemic hypoglycemia in man. It is still unclear whether loss of glucose homeostasis in these patients (partly) results from a dysregulation of beta cells. This study examines HADHSC expression in purified rat beta cells and investigates whether its selective suppression elevates insulin release. Beta cells expressed the highest levels of HADHSC mRNA and protein of all examined tissues, including those with high rates of mitochondrial beta-oxidation. On the other hand, beta cells expressed relatively low levels of other beta-oxidation enzymes (acyl-CoA dehydrogenase short, medium, and long chain and acetyl-coenzyme A acyltransferase 2). HADHSC expression was sequence-specifically silenced by RNA interference, and the effects were examined on glucose-stimulated insulin secretion following 48-72 h of suppression. In both rat beta cells and in the beta cell line INS1 832-13, HADHSC silencing resulted in elevated insulin release at low and at high glucose concentrations, which appeared not to be caused by increased rates of glucose metabolism or an inhibition in fatty acid oxidation. These data indicate that the normal beta cell phenotype is characterized by a high expression of HADHSC and a low expression of other beta-oxidation enzymes. Down-regulation of HADHSC causes an elevated secretory activity suggesting that this enzyme protects against inappropriately high insulin levels and hypoglycemia.     

Evidence that increased 12-lipoxygenase expression impairs pancreatic beta cell function and viability

Leukocyte type 12-lipoxygenase (12-LO) is an enzyme specifically expressed in the beta cells of the pancreas. 12-LO oxidizes fatty acids such as arachidonic acid and linoleic acids to their respective hydroperoxides. Increased concentration of lipid hydroperoxides causes oxidative stress and this could lead to cellular dysfunction. Increased expression of 12-LO in beta cells has been observed with use of inflammatory cytokines and during the prediabetic phase of beta cell dysfunction in the Zucker diabetic fatty rat model. Also mice deficient in 12-LO expression show a decreased incidence of immune-mediated diabetes. To further understand the role of 12-LO in beta cell metabolism, we over-expressed mouse leukocyte type 12-LO in INS-1 cells (transformed rat beta cell line) using an adeno-associated virus (AAV) vector system. In 12-LO over-expressing cells, cell-associated 12-HETE levels increased approximately 5- and approximately 3-fold in the culture supernatant. In the cells over-expressing 12-LO, glucose-stimulated insulin secretion (GSIS) decreased by 25-30% one hour after exposure to high glucose (15mM). By 2h, GSIS decreased by 50-54% at high glucose levels. These data suggest that increased 12-LO products can reduce the synthesis, processing or secretion of insulin in beta cells. We next examined the effect of 12-LO over-expression on mitogen-activated protein kinases (MAPK) by Western blot analyses using antibodies specific for different phospho-MAP kinases. Over-expression of 12-LO led to an activation of c-Jun N-terminal kinase while it markedly reduced Erk1 and 2 phosphorylation (4-fold). Further, over-expression of 12-LO led to induction of apoptosis in beta cells as determined by DNA ladder assay. These results suggest that increased 12-LO plays a key role in altering beta cell metabolism. Thus, increased 12-LO expression can have a detrimental effect on pancreatic beta cell function and viability, suggesting that blockade of 12-LO activity or expression could provide a novel way to protect beta cells from inflammatory injury.     

Differentiation of Y79 cells induced by prolonged exposure to insulin

Y79 human retinoblastoma cells are known to contain receptors for both insulin and insulin-like growth factors (IGFs), to produce these cytokines and release them in the culture medium. Previously we have demonstrated that IGFs and insulin stimulate Y79 cell proliferation through the involvement of type I IGF receptor and Insulin Receptor Substrate 1 (IRS-1). This paper studies the effect of prolonged exposure to insulin on Y79 cells. Cells grown for 10 days in the presence of insulin were reseeded and incubated once more with insulin. In the reseeded cells proliferation lowered and morphological changes appeared. After 10 days of reseeding, cells stopped proliferating and showed long ramifying neurite processes and varicosities consistent with neuronal differentiation. Morphological differentiation was accompanied by a marked increase in the content of total protein and in that of tubulin, the major protein constituent of microtubules, a marked increase in the content of specialized protein markers of dopaminergic and cholinergic differentiation (dopamine -hydroxylase and choline acetyltransferase activities, respectively); a contemporaneous decrease in the content of glial fibrillary acidic protein (GFAP), a specific marker of glial cells, was also observed. Our results demonstrate that prolonged exposure to insulin induces Y79 cells to differentiate into a neuronal-like phenotype. At this moment it is not possible to establish the mechanism by which insulin induces this differentiative effect.     

Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin

One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The glucose-lowering effect of the treatment might play an important role in the regeneration process.     

Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.     

Somatostatin inhibits insulin and glucagon release by monolayer cell cultures of rat endocrine pancreas

Dihydrosomatostatin (0.001–1.0 ug/ml) inhibited both insulin and glucagon secretion by monolayer cell cultures of newborn rat pancreas. When cultures were incubated with somatostatin and then rinsed, the effect of somatostatin appeared to last longer on the pancreatic alpha cell than on the beta cell as indicated by a more prolonged inhibition of glucagon secretion than of insulin release. Submaximal inhibition of glucose-stimulated insulin release by somatostatin was partially reversed by increasing the concentration of glucose. We conclude that the effect of somatostatin appears to be mediated directly on the pancreatic endocrine cells.     

Effect of jejunoileal bypass in the rat on the enteroinsular axis

The effect of jejunoileal bypass (JIB) on the enteroinsular axis was studied in vivo and in vitro in the rat. Glucose, insulin and GIP responses to oral glucose were compared in JIB and control rats. The effect of glucose and GIP on insulin release from the isolated perfused pancreas of the same animals was investigated to determine if JIB altered the sensitivity of the beta cell. Immunocytochemical studies of gut and pancreas were also carried out. Glucose, insulin and GIP responses to a glucose load were blunted after JIB, although basal GIP levels were elevated in these animals. The insulin response of the perfused JIB pancreas to GIP was 70% reduced from controls although the insulin response to glucose appeared normal. The size and area of JIB islets were unchanged from controls as was the distribution of insulin, glucagon, somatostatin and pancreatic polypeptide. GIP immunoreactive cells were present in all regions of the intestine including the JIB blind loop. This study confirms the findings of others that a relationship exists between reduced GIP and insulin response to oral glucose after JIB, and indicates that a decrease in sensitivity of the beta cell to GIP occurs following JIB that is not rapidly reversible. GIP secreted from blind loop mucosa may contribute to the high basal GIP found in JIB rats and may be causally connected to the fall in beta cell sensitivity.     

Effect of testosterone and 17beta-estradiol treatment on sex steroid binding proteins in the male of the green frog Rana esculenta

In this paper we report the effect of gonadectomy and/or long-term sex steroid (testosterone and estradiol-17beta) treatment and prolonged captivity (two months) on testosterone and estradiol-17beta binding proteins (TBP and EBP, respectively) in the plasma of the male of the green frog Rana esculenta. Experiments were carried out during different periods of the reproductive cycle. Gonadectomy and prolonged captivity were carried out in winter, when the spermatogenic activity slowed down and the concentration of circulating androgens was high. Both gonadectomy and prolonged captivity resulted in a significant decrease in TBP binding activity, which could not be restored by the hormonal treatment. On the contrary, when the hormonal treatment was carried out in the early summer, when the spermatogenesis was active but the concentration of circulating androgens was low, a significant increase in TBP binding activity was observed. Neither gonadectomy, nor the prolonged captivity, nor the hormonal treatment affected EBP levels. Our data indicate that TBP apparent changes in response to testosterone and estradiol-17beta treatment varied according to the period of the reproductive cycle, an indication that studies on sex steroid binding proteins regulation should take into consideration the internal endocrine condition before drawing any final conclusion especially in species with a seasonal mode of reproduction.     

Investigation of the effects of sulfonylurea exposure on pancreatic beta cell metabolism

Prolonged exposure of pancreatic beta cells to the sulfonylureas glibencamide and tolbutamide induces subsequent desensitization to the actions of these drugs. The precise mechanisms underlying this desensitization remain unknown, prompting the present study, which investigated the impact of prolonged sulfonylurea exposure on glucose and energy metabolism using clonal pancreatic BRIN-BD11 beta cells. Following prolonged exposure to tolbutamide, BRIN-BD11 beta cells were incubated in the presence of [U-(13)C]glucose, and isotopomer analysis revealed that there was a change in the ratio of flux through pyruvate carboxylase (EC 6.4.1.1) and pyruvate dehydrogenase (EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4). Energy status in intact BRIN-BD11 cells was determined using (31)P-NMR spectroscopy. Exposure to tolbutamide did not alter the nucleotide triphosphate levels. Collectively, data from the present study demonstrate that prolonged exposure of beta cells to tolbutamide results in changes in flux through key enzymes involved in glucose metabolism that, in turn, may impact on glucose-induced insulin secretion.     

Insulin increases H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced pancreatic beta cell death

Insulin resistance results, in part, from impaired insulin signaling in insulin target tissues. Consequently, increased levels of insulin are necessary to control plasma glucose levels. The effects of elevated insulin levels on pancreatic beta (β) cell function, however, are unclear. In this study, we investigated the possibility that insulin may influence survival of pancreatic β cells. Studies were conducted on RINm, RINm5F and Min-6 pancreatic β-cells. Cell death was induced by treatment with H₂O₂, and was estimated by measurements of LDH levels, viability assay (Cell-Titer Blue), propidium iodide staining and FACS analysis, and mitochondrial membrane potential (JC-1). In addition, levels of cleaved caspase-3 and caspase activity were determined. Treatment with H₂O₂ increased cell death; this effect was increased by simultaneous treatment of cells with insulin. Insulin treatment alone caused a slight increase in cell death. Inhibition of caspase-3 reduced the effect of insulin to increase H₂O₂-induced cell death. Insulin increased ROS production by pancreatic β cells and increased the effect of H₂O₂. These effects were increased by inhibition of IR signaling, indicative of an effect independent of the IR cascade. We conclude that elevated levels of insulin may act to exacerbate cell death induced by H₂O₂ and, perhaps, other inducers of apoptosis.     

Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in β cells to affect insulin secretion.     

Apolipoprotein A-I primes beta cells to increase glucose stimulated insulin secretion

The increase of plasma levels of high-density lipoproteins and Apolipoprotein A-I (ApoA-I), its main protein component, has been shown to have a positive action on glucose disposal in type 2 diabetic patients. The current study investigates the unexplored function of ApoA-I to prime beta cells for improved insulin secretion.INS-1E rat clonal beta cells as well as isolated murine islets were used to study the effect of ApoA-I on responsiveness of the beta cells to high glucose challenge. Confocal and transmission electron microscopy were used to dissect ApoA-I mechanisms of action. Chemical endocytosis blockers were used to understand the role of ApoA-I internalization in mediating its positive effect.Pre-incubation of beta cells and isolated murine islets with ApoA-I augmented glucose stimulated insulin secretion. This effect appeared to be due to an increased reservoir of insulin granules at the cell membrane, as confirmed by confocal and transmission electron microscopy. Moreover, ApoA-I induced pancreatic and duodenal homeobox 1 (PDX1) shuttling from the cytoplasm to the nucleus, with the subsequent increase in the proinsulin processing enzyme protein convertase 1 (PC1/3). Finally, the blockade of ApoA-I endocytosis in beta cells resulted in a loss of ApoA-I positive action on insulin secretion.The proposed mechanisms of the phenomenon here described include ApoA-I internalization into beta cells, PDX1 nuclear translocation, and increased levels of proinsulin processing enzymes. Altogether, these events lead to an increased number of insulin granules.     

Effects of ultra-wideband electromagnetic pulses on pre-neoplastic mammary epithelial cell proliferation

Electromagnetic ultra-wideband pulses (UWB) or nanopulses, are generated by a wide range of electronic devices used in communications and radar technology. However, the specific effects of nanopulse exposure on cell growth and function have not been extensively investigated. Here, studies have been conducted to determine the effects of prolonged exposure to non-ionizing, low to moderate intensity nanopulses on the growth of pre-neoplastic CL-S1 mammary epithelial cells in vitro. Cells were grown in culture and maintained in serum-free defined medium containing 10 ng/ml EGF and 10 microg/ml insulin as comitogens. Studies showed that 0.25-3.0 h exposure to nanopulses of 18 kV/m field intensity, 1 kHz repetition rate and 10 ns pulse width had no effect on CL-S1 cell growth or viability during the subsequent 72-h culture period. However, exposure to similar nanopulses for prolonged periods of time (4-6 h) resulted in a significant increase in cell proliferation, as compared to untreated controls. Additional studies showed that nanopulse exposure enhanced CL-S1 cell growth when cells were maintained in media containing only EGF, but had no effect on cells maintained in defined media that were mitogen-free or containing only insulin. Studies also showed that the growth-promoting effects of nanopulse exposure were associated with a relatively large increase in intracellular levels of phospho-MEK1 (active) and phospho-ERK1/2 (active) in these cells. These findings demonstrate that prolonged exposure to moderate levels of UWB enhanced EGF-dependent mitogenesis, and that this growth-promoting effect appears to be mediated by enhanced activation of the mitogen-activated protein kinase (MAPK) signalling pathway in pre-neoplastic CL-S1 mammary epithelial cells.