Marcello Anello

Functional and morphological alterations of mitochondria in pancreatic beta cells from type 2 diabetic patients

Aims/hypothesisLittle information is available on the insulin release properties of pancreatic islets isolated from type 2 diabetic subjects. Since mitochondria represent the site where important metabolites that regulate insulin secretion are generated, we studied insulin release as well as mitochondrial function and morphology directly in pancreatic islets isolated from type 2 diabetic patients.MethodsIslets were prepared by collagenase digestion and density gradient purification, and insulin secretion in response to glucose and arginine was assessed by the batch incubation method. Adenine nucleotides, mitochondrial membrane potential, the expression of UCP-2, complex I and complex V of the respiratory chain, and nitrotyrosine levels were evaluated and correlated with insulin secretion.ResultsCompared to control islets, diabetic islets showed reduced insulin secretion in response to glucose, and this defect was associated with lower ATP levels, a lower ATP/ADP ratio and impaired hyperpolarization of the mitochondrial membrane. Increased protein expression of UCP-2, complex I and complex V of the respiratory chain, and a higher level of nitrotyrosine were also found in type 2 diabetic islets. Morphology studies showed that control and diabetic beta cells had a similar number of mitochondria; however, mitochondrial density volume was significantly higher in type 2 diabetic beta cells.Conclusions/interpretationIn pancreatic beta cells from type 2 diabetic subjects, the impaired secretory response to glucose is associated with a marked alteration of mitochondrial function and morphology. In particular, UCP-2 expression is increased (probably due to a condition of fuel overload), which leads to lower ATP, decreased ATP/ADP ratio, with consequent reduction of insulin release.

Cellular and molecular effects of protons: Apoptosis induction and potential implications for cancer therapy

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.

Fast reversibility of glucose-induced desensitization in rat pancreatic islets. Evidence for an involvement of ionic fluxes.

The present study was done to achieve a better understanding of the role of ionic flux alterations in glucose-induced desensitization of pancreatic β-cells. Moreover, we investigated the reversibility of glucose-induced desensitization after different times of exposure to high glucose to ascertain the time necessary for desensitization reversal and to determine whether it depends on the length of high glucose exposure. Glucose desensitization was obtained by incubating rat pancreatic islets for 6 h in CMRL medium containing 16.7 mmol/l glucose. At the end of this period, insulin release, 86Rb efflux, and 45Ca uptake were measured in parallel experiments. In islets cultured at 16.7 mmol/l glucose, maximal glucose-induced insulin release was reduced (848 ± 97 pg · islet™1 · 30 min−1) in comparison to islets incubated at 5.5 mmol/l glucose (1,436 ± 144, n = 7, P < 0.01). In contrast, insulin content was similar in the two groups, being 41.0 ± 2.7 and 47.8 ± 2.2 ng/islet in islets exposed to 16.7 or 5.5 mmol/l glucose, respectively (P = 0.167). The effect of glucose on both 86Rb efflux and 45Ca uptake was also significantly reduced in 16.7 mmol/l glucose-cultured islets. 86Rb efflux was inhibited only 19 ± 4% in islets cultured at high glucose with respect to 56 ± 7% in control islets (n = 5, P < 0.001). 45Ca uptake was 10.5 ± 1.7 pmol/islet (mean ± SE, n = 9) in islets cultured at high glucose with respect to 19.7 ± 2.4 pmol/islet in control islets (P < 0.001). In contrast, the effect of glyburide (10 μmol/l) on insulin release, 86Rb efflux, and 45Ca uptake was similar in islets exposed to 5.5 or 16.7 mmol/l glucose. All the abnormalities observed in islets cultured at 16.7 mmol/l glucose were promptly and simultaneously reversible after islets were transferred in culture medium at 5.5 mmol/l glucose; both insulin secretion and glucose effects on 86Rb efflux and 45Ca uptake returned to values similar to control islets within 5 min. Also, islets exposed to high glucose for a longer period (24 h) recovered from both secretory and ionic abnormalities after 5 min of incubation in CMRL medium at 5.5 mmol/l glucose. Reversal from glucose desensitization was slower (45–60 min) when islets were incubated at 5.5 mmol/l glucose in Krebs-Ringer HEPES buffer instead of CMRL medium. The present data suggest that ion flux and consequent membrane-potential changes play a key role in the mechanism leading to glucose-induced desensitization of pancreatic β-cells. Because a normal response to glyburide was observed in islets exposed to high glucose, a proximal signal defect for closure of K+ channels rather than an intrinsic defect in the channel is likely.

Effects of prolonged glucose stimulation on pancreatic beta cells: from increased sensitivity to desensitization.

The prolonged exposure of pancreatic islets and isolated beta cells to elevated glucose concentrations induces a state of unresponsiveness to glucose (desensitization). However, an increased sensitivity to glucose (detected by a shift to the left of the dose-response curve of glucose-induced insulin release) has been also reported after chronic exposure to glucose, making the overall response less comprehensible. In vitro models have many theoretical and practical advantages in better understanding the effects of the prolonged glucose stimulation; moreover, they are also suitable for studying the mechanisms responsible of the observed alterations. We have performed a time-course study of the effect of the exposure to glucose at high concentration on the secretory behaviour of beta cells. Rat pancreatic islets exposed for 30 min to high glucose (300 mg/dl) showed increased basal insulin secretion (175±29 vs 44±8 pg/islet (per 30 min;n=5,P<0.002) as the only difference from control islets (exposed to 100 mg/dl). After 3 h exposure to high glucose, also increased sensitivity to glucose was observed, as indicated by a shift to the left of the glucose dose-response curve (EC50 123±10 and 177±11 mg/dl, respectively;n=5,P<0.05). After 6 h exposure to high glucose, besides the two alterations already described, also a decrease in glucose-induced insulin release was observed (688±104 vs 1184±34 pg/islet per 30 min;n=5,P<0.01). We studied the mechanism responsible for these alterations and we found that the “supersensitivity” to glucose may be related to alterations in the “glucose-sensing” mechanism of beta cells, in particular in glucose phosphorylation. In contrast, in islets desensitized to glucose our data suggest that ion flux and consequent membrane potential changes play a key role in determining the secretory defect. Since a normal response to glyburide was observed, a proximal signal defect for closure of potassium channels is more likely than an intrinsic defect in the channel. In conclusion, our data show what the prolonged stimulation of beta cells with glucose at high concentration induces a series of distinct secretory abnormalities, with a pattern of response that leads first to increased sensitivity and then to decreased responsiveness to glucose.

Exposure to glibenclamide increases rat beta cells sensitivity to glucose

An increased sensitivity to glucose was observed in islets pre‐exposed for 1 h to glibenclamide (0.1 μmol 1−1), but not to tolbutamide (100 μmol l−1), as indicated by a shift to the left of the dose‐response curve (EC50 at 5.8±0.3 mmol l−1 glucose vs 10.6±0.8 in control islets; n=11, P<0.005). According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l−1 glucose was higher in islets exposed to glibenclamide. Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion‐enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124±7 vs 51±9 nmol μg prot−1 90 min−1, P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar. When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l−1 dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC50 10.9±1.3 mmol l−1 glucose, n=3, similar to that of control islets). These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial‐bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug.

Hexokinase Shift to Mitochondria Is Associated With an Increased Sensitivity to Glucose in Rat Pancreatic Islets

When rat pancreatic islets are incubated in 5.5 or 16.7 mmol/1 glucose for 3 h, an increased sensitivity is observed in islets pre-exposed to high glucose, as indicated by a shift to the left of the glucose dose-response curve (EC50 7.1 ± 0.9 and 11.5 ± 1.2 in high- and low-glucose-exposed islets, respectively; n = 5, P < 0.05). To investigate the mechanism(s) responsible for this effect, we measured hexokinase and glucokinase activity both in the cytosolic fraction and in a mitochondrion-enriched fraction, since binding to the outer mitochondrial membrane has been reported to result in an increased enzyme activity. In islets cultured at 16.7 mmol/1 glucose, the cytosolic hexokinase activity was similar to control islets, but mitochondrial enzyme activity was significantly increased (124 ± 7 vs. 51 ± 9 nmol · µg−1 · 90 min−1, P < 0.01). As a consequence, the cytosolicrmitochondrial fraction ratio was altered in comparison with control islets. In contrast, glucokinase activity in the two groups of islets was similar in the cytosolic fraction and undetectable in the mitochodrial fraction. Hexokinase I quantitation by Western blot confirmed the enzyme translocation from the free cytosolic to the mitochondria-bound form in islets cultured at 16.7 mmol/l glucose. Glucose-induced alterations were reversible after 1 h exposure to 5.5 mmol/l glucose. Moreover, in islets exposed to 16.7 mmol/l glucose, inhibition of hexokinase binding to mitochondria by the addition of 20 nmol/1 dicyclohexylcarbodiimide resulted in no increase of glucose sensitivity (EC50 10.9 ± 0.4, n = 3, similar to that of control islets). These data indicate that after chronic exposure to high glucose, the β-cell becomes more sensitive to glucose before eventually getting desensitized. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity secondary to a subcellular shift of the enzyme from the free cytosolic to the mitochondriabound, more active form.

Interleukin-1β inhibition of insulin release in rat pancreatic islets: Possible involvement of G-proteins in the signal transduction pathway

SummaryIn vitro exposure of rat pancreatic beta cells to interleukin-1β (IL-lβ) inhibits glucose-stimulated insulin release (2140 α 239 and 323±80 pg · islet-1 · h-1 at glucose levels of 16.7 mmol/l in control and IL-l β-exposed islets, respectively, n = 7, p < 0.001). Cholera toxin (2 ηg/ml) or pertussis toxin (0.5 ηg/ml) potentiated, as expected, glucose-induced insulin release in control islets, but, in addition, when added together with IL-l β, were able to prevent the IL-l β mediated inhibition of glucose-stimulated insulin secretion (2087±301 and 1662±173 pg · islet-1 · h-1, respectively, p < 0.05 vs islets exposed to IL-l β alone). To investigate the mechanism by which the toxins prevent the IL-l β effect, we then measured nitrite levels, glucose oxidation and Ca2+ uptake. Nitrite levels in the culture medium were 4.2±1.4 and 24.0 ± 5 pmol · islet-1 · 24 h-1 in control islets and in IL-l β-exposed islets, respectively (n = 6, p = 0.05). In islets exposed to IL-l β and cholera or pertussis toxins, nitrite levels were 9.1±3 and 12.4 ± 6 pmol · islet-1 · 24 h-1, respectively (n = 6, NS vs control islets). Glucose oxidation at 16.7 mmol/l glucose was 31.1±2.9 pmol · islet-1 · 120 min-1 in control islets and 16.8 ± 2.7 pmol · islet-1 · 120 min-1 in IL-l β-treated islets (p < 0.05). The addition of cholera or pertussis toxins simultaneously to IL-l β prevented the inhibition of glucose oxidation at 16.7 mmol/l glucose (32.9±3.8 and 31.7±3.3 pmol · islet-1 · 120 min-1 in the presence of cholera or pertussis toxins, respectively). Glucose-stimulated 45Ca2+ uptake was also significantly inhibited in IL-l β-treated islets when compared to control islets (7.1±0.9 and 16.8±3.2 pmol · islet-1 · 20 min-1, respectively, p < 0.05). This inhibition was prevented by the presence of cholera or pertussis toxins (14.0±3.8 and 11.2±2.7 pmol · islet-1 · 20 min-1, respectively). In conclusion, our data show that cholera and, to a lesser extent, pertussis toxins are able to partially prevent the IL-lβ-induced increase in nitrite levels and block the inhibitory effects of IL-lβ on different steps leading to glucose-induced insulin secretion. These findings support the possibility that in pancreatic beta cells, G-proteins may be involved or interfere with the cytokine signal transduction.

Inhibition of the high-affinity glucose transporter GLUT 1 affects the sensitivity to glucose in a hamster-derived pancreatic beta cell line (HIT)

SummaryHIT is a hamster-derived beta-cell line which in contrast to normal beta cells that only express the high Km GLUT-2 glucose transporter, also expresses the low Km glucose transporter GLUT 1. In HIT cells the abnormal glucose transport mechanism is associated with a marked shift to the left of the glucose-induced insulin release dose-response curve. We have used this cell model to investigate whether changes in glucose transport affect the glucose-induced insulin release. HIT cells were first incubated with a concentration of cytochalasin B (0.4 μmol/l) that selectively inhibits the GLUT-1 but not the GLUT-2 transporter. The consequences of blocking glucose phosphorylation and insulin release were studied. Exposure to 0.4 μmol/l cytochalasin B for 1 h caused a selective loss of the low Km transport: the calculated Vmax of GLUT 1 was reduced from 1726±98 to 184±14 pmol · mg protein−1 5 min−1 (mean±SEM, n=6, p<0.005), while no major difference in the high Km (GLUT-2) transport was observed. In cytochalasin B exposed HIT cells the glucose phosphorylating activity (due to hexokinase and glucokinase) was unaffected. In these cells, however, the dose-response curve of glucose-induced insulin release was significantly shifted to the right: the 50% of maximal response (increment over baseline) was reached at an average glucose concentration of 2.9±0.2 mmol/l (vs 0.6±0.01 mmol/l in control HIT cells mean±SE, n=5, p<0.05) and the maximal effect was reached at 11.0 mmol/l glucose (vs 2.8 mmol/l in control HIT cells p<0.005). These results are consistent with the hypothesis that the affinity of the glucose transport system may contribute to determination of the glucose threshold concentration that triggers insulin secretion.

Glucotoxicity and lipotoxicity in the beta cell

Abstract In pancreatic islets chronically cultured with high glucose and free fatty acids (FFA), glucose-induced insulin secretion is impaired. Molecular mechanisms of this defect are still unclear. This article will focus on data showing the mechanisms by which chronically elevated levels of glucose and fatty acids may impair beta-cell function and, at a later stage, also affect beta-cell survival. Exposure of pancreatic islets to high glucose or FFA for 72 h impaired ATP production. This is associated to both decrease of glucose-induced pyruvate dehydrogenase (PDH) activity and increased UCP-2 expression. These data could indicate that both a defect in substrate supply to the Krebs cycle and an increase of energy disposal by increased UPC-2 protein expression could contribute to the reduced ATP production. When pancreatic islets were cultured with high levels of either FFA or glucose, or both, for 7 days, beta-cell apoptosis was increased. The nicotinamide prevents this effect, suggesting that oxidative stress could be involved in beta-cell damage. These findings seem to indicate that prolonged exposure to high glucose and FFA affects beta-cell function and, at later stage, destruction.