Effect of pentagastrin on IL-11β5 induced inhibition of insulin secretion inneonatal rat islets of Langerhans

AIM To obserwe tb e effect of pentagastrin (G-5) on IL-1β induced inhibition of insulin secretion in newbornrat islet of Langerhans.METHODS Islets of Langerhans of 3- to 5-day-old rats were isolated by collagenase digestion. The isletswere maintained free-floating in culture medium RPMI-1640, containing 10% (V/V) calf serum, anddistributed randomly in 96-well plastic plates (6 wells in each group). There are 15 islets per well in 0.2 mLculture medium. The islets were kept at 37℃ in mixed gases of 5% CO2 and 95% humidified air for the timerequired by the experimental design. Three experiments were performed in this study. (①) IL-13 inducedinhibition of insulin secretion in isolated islets of Langerhans. (②) Effect of G-5 on IL-lβ induced inhibition ofinsulin secretion. ③ Effect of G-5 on the functional repair of islet B-cells inhibited by IL-1β. Accumulatedand glucose stimulated insulin secretion was measured by radioimmunoassay in all studies. Data are presentedas ~ ± s. Differences between groups were analyzed using the Student's t test. P <0.05 was consideredsignificant.RESULTS The function of islet B-cells, which has been received 1L-1β treated for 24 hours, was dose-dependently inhibited. The accumulated and glucose stimulated insulin secretion was significantly lower thanthat of the control group (P<0. 05). The inhibitory effect of IL-1β on islet B-cells can be partially reversedby G-5. Accumulated and stimulated insulin secretion of G-5 0.6 ng/mL and 0.8 ng/mL groups wassignificantly higher than that of IL-1β treated alone group (P<0.05). The function of islet B-cells, whichreceived IL-iβ treatment for 24 hours, could partially recover after G-5 treatment for another 24 hours. Butaccumulated and glucose-stimulated insulin secretion in groups with G-5 treatment for 10 hours groups had nosignificant difference as compared with IL-1β treated alone group (P>0.05).CONCLUSION The present results indicate that G-5 may have a protective effect against the toxicity of IL-1β on islet B-cells.     

Different effects of glucose and glyburide on insulin secretion in rat pancreatic islets pre-exposed to interleukin-1β. Possible involvement of K+ and Ca2+ channels

Summary In vitro islet exposure to interleukin 1 inhibits the beta-cell response to glucose. We have studied whether a similar inhibition also occurs in response to the sulphonylurea glyburide. Rat pancreatic islets were cultured for 24 h in the presence or absence of 50 U/ml interleukin 1 and then stimulated with either glucose or glyburide for 1 h at 37 °C. In control islets basal insulin secretion was 117±32 pg · islet^–1 · h^–1 (mean ± SEM, n=7) and greatly increased in response to 16.7 mmol/l glucose (2140±293) or 10 mol/l glyburide (1464±234). When islets were pre-exposed to interleukin 1, insulin release was significantly reduced in response to glucose (323±80, p<0.001) but not in response to glyburide (1316±185). Since both glucose and glyburide influence beta-cell K⁺ and Ca²⁺ efflux, to further investigate this different response in islets exposed to interleukin 1 we measured both Rb⁺ efflux (as index of the ATP-sensitive K⁺ channel activity) and Ca²⁺ uptake. In control islets, the increased insulin secretion in response to 16.7 mmol/l glucose or 10 mol/l glyburide was associated with a reduction of ⁸⁶Rb efflux (decrement of –50±1.2 % and –49±2.3 %, respectively, mean ± SEM, n=5). In contrast, in interleukin 1pre-exposed islets both glucose and glyburide stimulation only slightly modified ⁸⁶Rb efflux (decrement of –19±1.9% and –5.3±3.1 %, respectively, n=5, p<0.001). ⁴⁵Ca²⁺ uptake in control islets was 2.6±0.4 pmol · islet^–1 · 20 min^–1 under basal conditions (at 2.8 mmol/l glucose), and increased to 16.8±3.2 and 10.7±2.1 pmol · islet^–1 · 20 min^–1 in islets stimulated with 16.7 mmol/l glucose or 10 mol/l glyburide, respectively (mean ± SEM, n=6). ⁴⁵Ca²⁺ uptake in interleukin 1 treated islets was higher than in control islets under basal conditions (4.6±0.6 pmol · islet^–1 · 20 min^–1 at 2.8 mmol/l glucose, p<0.05), but was significantly reduced in response to glucose 16.7 mmol/l (7.1±1.1, p<0.01 with respect to control islets). In contrast to glucose, 10 mol/l glyburide was able to stimulate calcium uptake in interleukin 1 treated islets in a similar way to control islets (12.8±2.5). The present data demonstrate that rat pancreatic islets treated with interleukin 1 for 24 h lose their responsivity to glucose, but not to glyburide. The difference between the two secretagogues is associated with the persistent ability of glyburide to influence Ca²⁺ uptake even in islets with impaired K⁺-channel function.     

Palmitate dependence of insulin secretion, “de novo” phospholipid synthesis and 45Ca2+-turnover in glucose stimulated rat islets

Summary Palmitate ability to modify D-[U-¹⁴C]glucose incorporation into different lipids (de novo synthesis), as well as sugar-stimulation of insulin release and ⁴⁵Ca²⁺-fluxes, was investigated in islets of fed and 48-h starved rats. The fatty-acid induced dose-dependent, correlative increments of insulin secretion, ⁴⁵Ca²⁺-influx and the de novo synthesis of each phospholipid fraction analysed at 20 mmol/l (but not 3 mmol/l) glucose. Omission of calcium reduced drastically (p<0.001) insulin release and the de novo synthesis of neutral glycerolipids, leaving unaltered that of acidic phospholipids (phosphatidate and phosphoinositides). The increased synthesis of the latter is therefore not the consequence of stimulated secretion. It could initiate or contribute to maintain an increased turnover of islet phosphoinositides, thus generating some mediators of the calcium signalling system (inositol phosphates). Starvation led to a drastic reduction (p<0.001) of both insulin secretion, de novo synthesis of each lipid fraction, and ⁴⁵Ca²⁺-influx in response to glucose and palmitate. The presence of a fatty-acid oxidation inhibitor (2-bromostearate or 2-tetradecylglycidate) prevented the effect of starvation on ⁴⁵Ca²⁺-influx, as it has been shown to do on insulin secretion and palmitate incorporation into islet lipids. It is finally suggested that palmitate might amplify the insulin secretory response of islets to glucose, through the stimulation of the de novo synthesis of phosphoinositides and the subsequent generation of inositol phosphates, which would contribute to accelerated calcium turnover.     

Functional identification and monitoring of individual α and β cells in cultured mouse islets of Langerhans

Abstract The aim of the present study was to evaluate, by use of fluorescence microscopy and immunofluorescence stainings, the use of a fluorescent membrane potential sensitive probe as a means to identify and monitor changes in membrane potential of individual cell types in whole islets of Langerhans over time. Our work supports the use of the fluorescent probe bis-(1,3 dibutylbarbituric acid) trimethine oxonol (diBAC₄(3)), in identification of single and cells in the periphery of mouse pancreatic islets cultured on extracellular matrix. At a low extracellular glucose concentration (3 mM), heterogeneous staining of the islets was observed. Approximately 97% of the peripheral cells that stained brightly with diBAC₄(3) were glucagon positive. Additional diBAC₄(3) studies, demonstrated that an increase in glucose concentration from 3 to 10 mM is paralleled by repolarization of cells and depolarization of cells. This suggests that reciprocity of glucagon and insulin release upon glucose stimulation is coupled to divergent changes in membrane potential of these cell types and supports the use of diBAC₄(3) as a means to detect changes in secretion in both cell types.     

Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.     

Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2’-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2’-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2’-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.     

IL-1β induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1beta (IL-1beta) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1beta exposed Wistar Furth (WF) rat islets. METHODS: The 82 protein spots, that changed expression after IL-1beta exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification. RESULTS: Forty-five different proteins were identified from 51 spots and grouped according to function: (i) energy transduction and redox potentials; (ii) glycolytic and Krebs cycle enzymes; (iii) protein, DNA and RNA synthesis, chaperoning and protein folding; (iv) signal transduction, regulation, differentiation and apoptosis; (v) cellular defence; and (vi) other functions. Comparison of IL-1beta exposed BB-DP and WF islets showed common changes in 14 proteins and several proteins influencing similar pathways, suggesting that similar routes in the two strains lead to beta-cell destruction. CONCLUSION/INTERPRETATION: We demonstrate that proteome analysis is a powerful tool to identify proteins and pathways in BB-DP rat islets exposed to IL-1beta.     

TNFα and IFNγ potentiate IL-1β induced mitogen activated protein kinase activity in rat pancreatic islets of Langerhans

Aims/hypothesis. Interleukin-1 beta (IL-1β) in synergy with tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) is cytotoxic to pancreatic beta cells. Mitogen-activated protein kinase (MAPK) activity that is induced by interleukin-1 beta has been suggested to signal nitric oxide-dependent as well as nitric oxide-independent beta-cell destructive pathways. The aim of this study was to investigate if TNFα and IFNγ signal through mitogen-activated protein kinases in isolated rat islets of Langerhans and if they potentiate mitogen-activated protein kinase activity induced by IL-1β.¶Methods. Islets of Langerhans were isolated from 5- to 7-day-old Wistar rats and precultured for 7 days before stimulation with IL-1β, TNFα and/or IFNγ for 20 min followed by lysis. Kinase activity was measured with a whole cell lysate kinase assay and after immunoprecipitation of the kinase using immunocomplex kinase assay.¶Results. Exposure to IL-1β or TNFα significantly increased mitogen-activated protein kinase activity, whereas IFNγ tended to decrease extracellular-signal-regulated kinase activity. Further, TNFα and IFNγ were found to synergistically increase mitogen-activated protein kinase activity induced by IL-1β.¶Conclusion/interpretation. We hypothesise that the synergistic effect of IL-1β, TNFα and IFNγ in the functional inhibition and induction of cell death in pancreatic beta cells is signalled through a synergistic activation of mitogen-activated protein kinase activity [Diabetologia (2000) 43: 1389–1396].     

Functional and morphological effects of interleukin-1β on the perfused rat pancreas

Summary We recently reported a potentiating effect of recombinant human interleukin-1 on glucose-stimulated insulin release from the isolated perfused pancreas. With the aim of determining whether the stimulatory effect of recombinant interleukin-1 on the B cell in the intact gland was modulated by varying the concentration, time of exposure to recombinant interleukin-1 or B-cell activity, and to elucidate a possible mechanism of action, we measured in the perfused rat pancreas the release of insulin, glucagon and/or prostaglandin E₂ according to the following three different protocols: (1) perfusion with 20 ng/ml of recombinant interleukin-1 for 92 min at 5 and 20 mmol/1 D-glucose (2) perfusion with varying concentrations of recombinant interleukin-1 ranging from 0.1×10^–3 ng/ml to 100 ng/ml at 5 and 20 mmol/l D-glucose (3) perfusion with 20 ng/ml of recombinant interleukin-1 at 5,11 or 20 mmol/l D-glucose. Furthermore, in a separate set of experiments we examined the influence of the cytokine on the morphology of the endocrine pancreas. Interleukin-1 stimulated insulin secretion at 11 and 20 mmol/l D-glucose and potentiated first as well as second phase insulin release in a dose-dependent fashion, with decreasing effect at higher concentrations. Glucagon secretion was also stimulated by recombinant interleukin-1, irrespective of increasing glucose (5, 11, 20 mmol/l) and insulin concentrations. The potentiating effect of recombinant interleukin-1 on insulin secretion was evident even after discontinued perfusion with the cytokine, suggesting a priming effect on B-cell function. Furthermore, we did not observe any relation between the recombinant interleukin-1 mediated insulin and glucagon release and prostaglandin E₂. Electron microscopy of the pancreata perfused with recombinant interleukin-1 revealed significant B cell and to a lesser extent A-cell lysis as well as induction of cell protrusions (blebs) in B cells only, accompanied by peripheral degranulation and rearrangement of rough endoplasmatic reticulum. We suggest that in addition to a paracrine effect of locally produced interleukin-1 systemic interleukin-1 may have an endocrine effect on A- and B-cell function and viability. Interleukin-1 should be considered to be a physiological modulator of insulin and glucagon secretion e.g. during the acute phase response, but also as a pathogenetic factor in Type 1 (insulin-dependent) diabetes mellitus.     

PAF effects on MCP-1 and IL-6 secretion in U-937 monocytes in comparison with oxLDL and IL-1β effects

Objective

To study the effects of PAF, in comparison with OxLDL and IL-1β on MCP-1 and IL-6 secretion from U-937 monocytes and to investigate the mechanism of its action.

Methods

U-937 cell line was cultured in the presence or absence of PAF or OxLDL or IL-1β. Secretion of IL-6 and MCP-1 was measured by ELISA method, mRNA levels of MCP-1 and PAFR was measured using real-time PCR. In order to investigate the mechanism of mediator's action signal transduction appropriate inhibitors was used and oxidant status of cells by measurement the total cellular thiols content and glutathione was determined.

Results and conclusion

None of the tested mediators induced the secretion of IL-6. On the other hand PAF and OxLDL caused a short-term while IL-1β caused a long-term secretion and expression of MCP-1. Reduced total thiol levels and GSH/GSSG ratio indicate that the above mediators induce oxidative stress. The signal transduction of all mediators is mediated through G-proteins, protein kinases (PKC, serine–threonine kinase and tyrosine kinase) and NF-κB activation. In addition, PAF, OxLDL, IL-1β activates monocytes leading to increased PAF receptor mRNA levels. These results indicate that PAF and OxLDL, in a different pattern from that of IL-1β, regulate MCP-1 expression via pathways that involve changes in cell redox status.     

The role of interleukin-1β and extracellular signal-regulated kinase 1/2 in glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) is one of the important physiological characteristics of islet β cells, and extracellular-regulated protein kinase 1/2 (ERK1/2) is an important member of the mitogen-activated protein kinase family that regulates this process. The inflammatory cytokine interleukin (IL)-1β can inhibit the insulin secretion of pancreatic β cells, but the exact mechanism is unclear. This study was designed to investigate the inhibitory effect of IL-1β on GSIS in βTC-6 cells and its relation with the ERK1/2 signal transduction pathway. β-TC6 cells were cultured and stimulated with 0mM, 1.38mM, or 5.5mM glucose. In addition, GSIS in β-TC6 cells was blocked by IL-1β at concentrations of 0.15 ng/mL, 1.5 ng/mL, and 15 ng/mL. After glucose stimulation and IL-1β intervention, the insulin level in the cell supernatant was detected by radioimmunoassay, and the phosphorylation level of ERK1/2 was detected by western blotting assay. The insulin level in the 1.38mM glucose group was 108.52 ± 5.94 uIU/mL, which was significantly higher than the 0mM and 5.5mM glucose groups (p < 0.05). Compared with the 0mM glucose group, the level of ERK1/2 phosphorylation was increased in the 1.38mM and 5.5mM glucose groups. After intervention by 0.15 ng/mL, 1.5 ng/mL, and 15 ng/mL IL-1β, the level of ERK1/2 phosphorylation induced by 1.38mM glucose stimulation decreased in a dose-dependent manner, and the insulin level correspondingly decreased. IL-1β can inhibit GSIS in βTC-6 cells, which is related to its inhibition of the phosphorylation of ERK1/2.     

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia, suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared to controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2, in isolated islets, leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.     

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1^-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1^-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1^-/- islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1^-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1^-/- islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1^-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.     

Effect of troglitazone on insulin secretion in pancreatic β-cells and its mechanism

The effect of troglitazone on glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and its mechanism were investigated.10 μmol/L troglitazone had no effect on basal insulin secretion,but significantly decreased GSIS and stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylations (all P<0.01).These reactions were completely reversed by AMPK inhibitor compound C,suggesting that the troglitazone acutely inhibits insulin secretion via stimulating AMPK activity in beta cells.     

Effect of troglitazone on insulin secretion in pancreatic β-cells and its mechanism

The effect of troglitazone on glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and its mechanism were investigated.10 μmol/L troglitazone had no effect on basal insulin secretion,but significantly decreased GSIS and stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylations (all P<0.01).These reactions were completely reversed by AMPK inhibitor compound C,suggesting that the troglitazone acutely inhibits insulin secretion via stimulating AMPK activity in beta cells.     

Human interleukin-1β induced stimulation of insulin release from rat pancreatic islets is accompanied by an increase in mitochondrial oxidative events

Summary Acute exposure of pancreatic islets to interleukinl-1 results in an increase in insulin release, while an extension of the exposure time induces a functional suppression and eventually, destruction of the B-cells. We have recently suggested that the interleukin-1 induced inhibition of islet function is mediated through an impairment in oxidative metabolism. The aim of the current study was to investigate if the acute, stimulatory effects of interleukin-1 on islet function could also be related to changes in the substrate metabolism. For this purpose, rat islets were exposed for 90–120 min to 30 pmol/l human recombinant interleukin-1 (biological activity of 2.5 U/ml) and their function and metabolism characterized during this period. The cytokine did not increase insulin release in the presence of 1.7 or 5.5 mmol/l glucose but in both the presence of 16.7 mmol/l glucose or 10 mmol/l leucine + 2 mmol/l glutamine there was a 50% increase in insulin release. Interleukin-1 exposure increased the oxidation of D-[U-¹⁴C]glucose at 5.5 mmol/l glucose by 25% and at 16.7 mmol/l glucose by 60%. Carbohydrate and amino acid metabolism were further examined in the presence of D-[5-³H] glucose, D-[6-¹⁴C]glucose, [1-¹⁴C]pyruvate, L-[U-¹⁴C]glutamine, L-[U-¹⁴C]leucine and L-[1-¹⁴C]leucine. There was no difference between control islets and interleukin-1 exposed islets in terms of D-[5-³H]glucose utilization or [1-¹⁴C]pyruvate decarboxylation, but the oxidation of D-[6-¹⁴C]glucose was increased by 64% in the interleukin-1 exposed islets. There was also an interleukin-1 induced 45–60% increase in the decarboxylation of L-[1-¹⁴C]leucine and oxidation of L-[U-¹⁴C]leucine and L-[U-¹⁴C]glutamine, all intramitochrondrial events. The stimulation of insulin release by interleukin-1 in the presence of 16.7 mmol/l glucose was abolished in islets incubated in Ca²⁺ depleted medium, but the rate of D-[6-¹⁴C] glucose oxidation remained elevated (47% increase at 16.7 mmol/l glucose). These data indicate an increase in substrate metabolism at the mitochondrial level during acute exposure of rat pancreatic islets to interleukin-1. The increase in oxidative events can explain the observed interleukin-1 induced increase in insulin release during glucose stimulation. Furthermore, these findings raise the possibility that mitochondria are primary targets of interleukin-1 action in the B-cells.     

Physiological and biochemical effects of 17β estradiol in aging female rat brain

Aging in females and males is considered as the end of natural protection against age related diseases like osteoporosis, coronary heart disease, diabetes, Alzheimer's disease and Parkinson's disease. These changes increase during menopausal condition in females when the level of estradiol is decreased. The objective of this study was to observe the changes in activities of monoamine oxidase, glucose transporter-4 levels, membrane fluidity, lipid peroxidation levels and lipofuscin accumulation occurring in brains of female rats of 3 months (young), 12 months (adult) and 24 months (old) age groups, and to see whether these changes are restored to normal levels after exogenous administration of estradiol (0.1 μg/g body weight for 1 month). The results obtained in the present work revealed that normal aging was associated with significant increases in the activity of monoamine oxidase, lipid peroxidation levels and lipofuscin accumulation in the brains of aging female rats, and a decrease in glucose transporter-4 level and membrane fluidity. Our data showed that estradiol treatment significantly decreased monoamine oxidase activity, lipid peroxidation and lipofuscin accumulation in brain regions of aging rats, and a reversal of glucose transporter-4 levels and membrane fluidity was achieved, therefore it can be concluded from the present findings that estradiol's beneficial effects seemed to arise from its antilipofuscin, antioxidant and antilipidperoxidative effects, implying an overall anti-aging action. The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders.     

Association between islets of Langerhans and pancreatic ductal system in adult rat. Where endocrine and exocrine meet together?

Aims/hypothesis. Studies on the functional and morphological relations between exocrine and endocrine pancreas have been conducted mainly to disclose the influence of islets of Langerhans on acinar parenchyma. Less attention has been paid to the relations occurring between islets and pancreatic ducts. Methods. A series of consecutive sections of normal adult rat pancreas were double stained with islet (hormones) and duct (cytokeratin 20) markers. Electron microscopy was conducted to investigate the ultrastructural features of duct-islet relations and anti-insulin immunogold labelling was carried out to reveal the presence of insulin in the pancreatic duct system. Results. Consecutive double-stained sections demonstrated that 73.60 ± 2.97 % of the islets were attached to the ducts. For each series, 93.48 ± 5.43 % of the islets contacting the duct tree were associated with small-sized ducts or centroacinar cells. Electron microscopy revealed that some insulin and somatostatin cells do face the duct lumen. Insulin was detected within the duct lumen and in the endosomal compartment of the duct cells. Conclusions/interpretation. The finding that most islets are connected with the duct system in the adult pancreas is discussed in terms of hormone secretion into the ducts, islet histogenesis and the relation among the three tissue components of the pancreas, the endocrine, the exocrine and the duct system. [Diabetologia (2001) 44: 575–584] Received: 12 October 2000 and in revised form: 18 January 2001     

Dissociated effects of glucose-dependent insulinotropic polypeptide vs glucagon-like peptide-1 on β-cell secretion and insulin clearance in mice

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) potently augment insulin response to glucose. It is less known what their effects are insulin clearance, which also contributes to peripheral hyperinsulinemia observed after administration of incretins together with glucose. The aims of this study were the quantification of C-peptide secretion and the evaluation of insulin clearance after administration of GIP with glucose. This allows the assessment of GIP's effects on hyperinsulinemia. In addition, GIP's effects were compared with those of GLP-1. Anesthetized female NMRI mice were injected intravenously with glucose alone (1 g/kg, n = 35) or glucose together with GIP (50 μg/kg, n = 12). Samples were taken through the following 50 minutes, and C-peptide and insulin concentrations were used to reconstruct C-peptide secretion rate and insulin clearance. In a previous study, GLP-1 (10 μg/kg) was used in 12 mice; and we used those GLP-1 results to compare GIP effects with those of GLP-1. C-peptide secretion rate peaked at 1 minute after glucose injection, and the immediate part of the insulin-releasing process was markedly augmented by both incretin hormones (1-minute suprabasal increment secretory rate was 20 ± 2 pmol/min for GIP and 28 ± 2 pmol/min for GLP-1, vs only 9 ± 1 pmol/min for glucose alone; P < .001). Until 10 minutes after administration, C-peptide secretion remained higher with incretins (P < .0001), whereas starting from 20 minutes, the 3 patterns were undistinguishable (P > .2). Insulin clearance, previously shown to be abridged by 46% with GLP-1, was reduced only by a nonsignificant (P = .27) 21% with GIP. This study thus shows that the 2 incretins markedly augment glucose-stimulated insulin secretion in mice by a preferential action on the immediate response to glucose of insulin secretion. However, the action of GIP is less effective than that of GLP-1. Insulin clearance with GIP is not significantly reduced. We conclude that GIP is less potent than GLP-1 in inducing glucose-stimulated hyperinsulinemia in the mouse.     

Protein phosphatase 1 (PP-1)-dependent inhibition of insulin secretion by leptin in INS-1 pancreatic β-cells and human pancreatic islets

Leptin inhibits insulin secretion from pancreatic β-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic β-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 β-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on β-cell function was characterized by insulin-secretion assays, intracellular Ca2(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic β-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in β-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 β-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.