In an effort to better understand the phenomenon of lipotoxicity in human ␤-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of ␤-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and proapoptotic effects on human pancreatic ␤-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.

Aims/hypothesis: Little 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. Methods: Islets 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. Results: Compared 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/ interpretation: In 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.

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In rat pancreatic islets chronically exposed to high glucose or high free fatty acid (FFA) levels, glucoseinduced insulin release and mitochondrial glucose oxidation are impaired. These abnormalities are associated with high basal ATP levels but a decreased glucoseinduced ATP production (⌬ of increment over baseline 0.7 ؎ 0.5 or 0.5 ؎ 0.3 pmol/islet in islets exposed to glucose or FFA vs. 12.0 ؎ 0.6 in control islets, n ‫؍‬ 3; P < 0.01) and, as a consequence, with an altered ATP/ADP ratio. To investigate further the mechanism of the impaired ATP formation, we measured in rat pancreatic islets glucose-stimulated pyruvate dehydrogenase (PDH) activity, a key enzyme for pyruvate metabolism and for the subsequent glucose oxidation through the Krebs cycle, and also the uncoupling protein-2 (UCP-2) content by Western blot. In islets exposed to high glucose or FFA, glucose-stimulated PDH activity was impaired and UCP-2 was overexpressed. Because UCP-2 expression is modulated by a peroxisome proliferatoractivated receptor (PPAR)-dependent pathway, we measured PPAR-␥ contents by Western blot and the effects of a PPAR-␥ antagonist. PPAR-␥ levels were overexpressed in islets cultured with high FFA levels but unaffected in islets exposed to high glucose. In islets exposed to high FFA concentration, a PPAR-␥ antagonist was able to prevent UCP-2 overexpression and to restore insulin secretion and the ATP/ADP ratio. These data indicate that in rat pancreatic islets chronically exposed to high glucose or FFA, glucose-induced impairment of insulin secretion is associated with (and might be due to) altered mitochondrial function, which results in impaired glucose oxidation, overexpression of the UCP-2 protein, and a consequent decrease of ATP production. This alteration in FFA cultured islets is mediated by the PPAR-␥ pathway. Diabetes 51: 2749 -2756, 2002 P atients with type 2 diabetes are characterized by a progressive decline of insulin secretion that becomes more severe with the increasing duration of the disease (1-4). In these patients, the mechanisms that cause the progressive ␤-cell failure are currently under investigation: the altered insulin secretory pattern depends, at least in part, on the negative influence of chronic high glucose (5-8) and/or high free fatty acid (FFA) (9 -12) plasma concentrations (gluco-or lipotoxicity). These metabolites are believed to affect pancreatic ␤-cell function by chronic ␤-cell stimulation and consequent "desensitization" to glucose. However, the molecular mechanisms of glucose desensitization induced by hyperglycemia or hypernefemia are still unclear (13,14).Rat pancreatic islets that are chronically exposed to high glucose or FFAs have an impaired glucose oxidation (15). Because glucose oxidation generates ATP and the rise of ATP and ATP/ADP ratio plays a central role in glucose-induced insulin release by causing K ϩ -ATP channel closure, membrane depolarization, increased calcium influx, and insulin granule exocytosis, we first measured ATP and ADP levels in islets that were chronic...

Because metformin affects glucose and free fatty acid (FFA) metabolism in peripheral insulin target tissues, we investigated the effect of this drug in restoring a normal secretory pattern in rat pancreatic islets whose function has been impaired by chronic exposure to elevated FFA or glucose concentrations. We cultured rat pancreatic islets with or without FFA (2 mmol/l oleate/ palmitate 2:1) or high glucose (16.7 mmol/l) concentrations in the presence or absence of metformin (0.25-12.5 µg/ml) and then measured insulin release, glucose utilization, glucose, and FFA oxidation. When compared with control islets, islets exposed to high FFA or glucose concentrations showed an increased basal and a decreased glucose-induced insulin release. In islets cultured for an additional 24 h with FFA or glucose in the presence of metformin (2.5 µg/ml), both basal and glucose-induced insulin secretions were restored. Both glucose utilization and glucose oxidation were altered in islets pre-exposed to high FFA or glucose concentrations. In particular, regarding control islets, glucose utilization was increased at 2.8 mmol/l glucose and decreased at 16.7 mmol/l glucose; glucose oxidation was similar to control islets at 2.8 mmol/l glucose but decreased at 16.7 mmol/l glucose. In contrast, oleate oxidation was increased in islets pre-exposed to FFA. All of these abnormalities were reversed in islets cultured for an additional 24 h with high FFA or glucose concentrations in the presence of metformin (2.5 µg/ml). In conclusion, our data show that metformin is able to restore the intracellular abnormalities of glucose and FFA metabolism and to restore a normal secretory pattern in rat pancreatic islets whose secretory function has been impaired by chronic exposure to elevated FFA or glucose levels. These data raise the possibility that, in diabetic patients, metformin (in addition to its peripheral effects) may have a direct beneficial effect on the ␤-cell secretory function. Diabetes 49:735-740, 2000 T he biguanide metformin is widely used in Europe, Canada, and more recently the U.S. for the treatment of type 2 diabetic patients. Its antihyperglycemic effect is not a consequence of insulin secretion stimulation but rather is an effect on peripheral tissues that makes them more sensitive to insulin action. In vitro and in vivo studies indicate that the glucose-lowering effect of metformin is mainly the consequence of decreased hepatic glucose output and increased peripheral glucose uptake and utilization (1-8).At the cellular level, metformin has different effects on glucose and free fatty acid (FFA) metabolism. In muscle, adipose tissue, and liver, metformin increases glucose oxidation but decreases FFA oxidation (9,10). However, whether metformin also affects glucose and FFA oxidation in other tissues, including the pancreatic ␤-cells, is unknown. This may be a relevant issue because chronic elevation of glucose or FFAs is known to inhibit insulin secretion in isolated pancreatic islets (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21...

Type 2 (non-insulin-dependent) diabetes results from decreased insulin action in peripheral target tissues (insulin resistance) and impaired pancreatic ␤-cell function. These defects reflect both genetic components and environmental risk factors. Recently, the common Gly 972 3 Arg amino acid polymorphism of insulin receptor substrate 1 (Arg 972 IRS-1) has been associated with human type 2 diabetes. In this study, we report on some functional and morphological properties of isolated human islets carrying the Arg 972 IRS-1 polymorphism. Insulin content was lower in variant than control islets (94 ؎ 47 vs. 133 ؎ 56 U/islet; P < 0.05). Stepwise glucose increase (1.7 to 16.7 mmol/l) significantly potentiated insulin secretion from control islets, but not Arg 972 IRS-1 islets, with the latter also showing a relatively lower response to glyburide and a significantly higher response to arginine. Proinsulin release mirrored insulin secretion, and the insulin-to-proinsulin ratio in response to arginine was significantly lower from Arg 972 IRS-1 islets than from control islets. Glucose utilization and oxidation did not differ in variant and wild-type islets at both low and high glucose levels. Electron microscopy showed that Arg 972 IRS-1 ␤-cells had a severalfold greater number of immature secretory granules and a lower number of mature granules than control ␤-cells. In conclusion, Arg 972 IRS-1 islets have reduced insulin content, impaired insulin secretion, and a lower amount of mature secretory granules. These alterations may account for the increased predisposition to type 2 diabetes in individuals carrying the Gly 972 3 Arg amino acid polymorphism of IRS-1.

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.

1 To determine how pretreatment with sulphonylureas alters the b cell function, mouse islets were cultured (18 ± 20 h) without (controls) or with (test) 0.01 mM glibenclamide. Acute responses to glucose were then determined in the absence of glibenclamide. 2 Test islets were insensitive to drugs (sulphonylureas and diazoxide) acting on K + -ATP channels, and their [Ca 2+ ] i was already elevated in the absence of stimulation. 3 Insulin secretion was increased in the absence of glucose, and mainly stimulated between 0 ± 10 instead of 7 ± 20 mM glucose in controls. The maximum response was halved, but this di erence disappeared after correction for the 45% decrease in the islet insulin content. 4 The ®rst phase of glucose-induced insulin secretion was abrogated because of a paradoxical decrease of the high basal [Ca 2+ ] i in b cells. The second phase was preserved but occurred with little rise of [Ca 2+ ] i . These abnormalities did not result from alterations of glucose metabolism (NADPH uorescence). 5 In islets cultured with 50 mM tolbutamide, glucose induced biphasic increases in [Ca 2+ ] i and insulin secretion. The decrease in the secretory response was matched by the decrease in insulin content (45%) except at maximal glucose concentrations. Islets pretreated with tolbutamide, however, behaved like those cultured with glibenclamide if tolbutamide was also present during the acute functional tests. 6 In conclusion, treatment with a low glibenclamide concentration causes long-lasting blockade of K + -ATP channels and rise of [Ca 2+ ] i in b cells. Glucose-induced insulin secretion occurs at lower concentrations, is delayed and is largely mediated by a modulation of Ca 2+ action on exocytosis. It is suggested that glucose regulation of insulin secretion mainly depends on a K + -ATP channelindependent pathway during in vivo sulphonylurea treatment.