Oxygen-dependent Regulation of Erythropoietin Receptor Turnover and Signaling.

von Hippel-Lindau (VHL) disease is a rare familial cancer predisposition syndrome caused by a loss or mutation in a single gene,VHL, but it exhibits a wide phenotypic variability that can be categorized into distinct subtypes. The phenotypic variability has been largely argued to be attributable to the extent of deregulation of the α subunit of hypoxia-inducible factor α, a well established target of VHL E3 ubiquitin ligase, ECV (Elongins/Cul2/VHL). Here, we show that erythropoietin receptor (EPOR) is hydroxylated on proline 419 and 426 via prolyl hydroxylase 3. EPOR hydroxylation is required for binding to the ß domain of VHL and polyubiquitylation via ECV, leading to increased EPOR turnover. In addition, several type-specific VHL disease-causing mutants, including those that have retained proper binding and regulation of hypoxia-inducible factor α, showed a severe defect in binding prolyl hydroxylated EPOR peptides. These results identify EPOR as the secondbona fidehydroxylation-dependent substrate of VHL that potentially influences oxygen homeostasis and contributes to the complex genotype-phenotype correlation in VHL disease.

Thermogenic capacity is antagonistically regulated in classical brown and white subcutaneous fat depots by high fat diet and endurance training in rats: impact on whole-body energy expenditure.

This study investigated the regulation of thermogenic capacity in classical brown adipose tissue (BAT) and subcutaneous inguinal (SC Ing) white adipose tissue (WAT) and how it affects whole-body energy expenditure in sedentary and endurance-trained rats fed ad libitum either low fat or high fat (HF) diets. Analysis of tissue mass, PGC-1α and UCP-1 content, the presence of multilocular adipocytes, and palmitate oxidation revealed that a HF diet increased the thermogenic capacity of the interscapular and aortic brown adipose tissues, whereas exercise markedly suppressed it. Conversely, exercise induced browning of the SC Ing WAT. This effect was attenuated by a HF diet. Endurance training neither affected skeletal muscle FNDC5 content nor circulating irisin, but it increased FNDC5 content in SC Ing WAT. This suggests that locally produced FNDC5 rather than circulating irisin mediated the exercise-induced browning effect on this fat tissue. Importantly, despite reducing the thermogenic capacity of classical BAT, exercise increased whole-body energy expenditure during the dark cycle. Therefore, browning of subcutaneous WAT likely exerted a compensatory effect and raised whole-body energy expenditure in endurance-trained rats. Based on these novel findings, we propose that exercise-induced browning of the subcutaneous WAT provides an alternative mechanism that reduces thermogenic capacity in core areas and increases it in peripheral body regions. This could allow the organism to adjust its metabolic rate to accommodate diet-induced thermogenesis while simultaneously coping with the stress of chronically increased heat production through exercise.

Chronic AICAR-induced AMP-kinase activation regulates adipocyte lipolysis in a time-dependent and fat depot-specific manner in rats.

This study investigated the effects of chronic in vivo AMP-kinase activation with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) on lipolysis in subcutaneous inguinal, epididymal, and retroperitoneal fat pads. Male Wistar rats received daily single intraperitoneal injections of either saline or AICAR (0.7 g/kg body wt) for a period of 8 wk. The fat pads were used either to isolate adipocytes and measure basal and catecholamine-stimulated lipolysis or to assess signaling steps of lipolysis after 4 and 8 wk of AICAR treatment. Blood was sampled weekly to measure nonesterified fatty acids (NEFAs). AICAR treatment reduced basal and catecholamine-stimulated lipolysis at week 4 in adipocytes from all fat depots. However, at week 8, catecholamine-induced lipolysis significantly increased in inguinal and retroperitoneal adipocytes. Interestingly, plasma levels of NEFAs were also decreased and subsequently increased at 4 and 8 wk, respectively. The lipolytic cascade of the inguinal fat pad was the most drastically affected by the treatment, since the phosphorylation and content of most proteins involved in lipolysis were consistently undetected in this tissue after 4 and 8 wk of AICAR treatment. The enhancement of catecholamine-induced lipolysis in inguinal and retroperitoneal adipocytes after 8 wk of AICAR treatment was accompanied by increased contents of adipose triglyceride lipase (ATGL) and perilipin A in these fat depots. In summary, despite depot-specific regulation of the lipolytic cascade, catecholamine-induced lipolysis in isolated adipocytes correlated well with plasma NEFA concentrations in the course of chronic AICAR-induced AMPK activation. The mechanisms underlying these effects also involved time-dependent and depot-specific regulation of hormone-sensitive lipase, ATGL, and perilipin.

Distinct mechanisms involving diacylglycerol, ceramides, and inflammation underlie insulin resistance in oxidative and glycolytic muscles from high fat-fed rats.

This study investigated whether oxidative and glycolytic rat skeletal muscles respond differently to a high-fat (HF) sucrose-enriched diet with respect to diacylglycerol (DAG) and ceramides accumulation, protein kinase C (PKC) activation, glucose metabolism, and the expression of inflammatory genes. HF diet (8 weeks) suppressed insulin-stimulated glycogen synthesis and glucose oxidation in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. However, DAG and ceramides levels increased in Sol and EDL, but not in Epit muscles of HF-fed rats. Additionally, membrane-bound PKC-delta and PKC-theta increased in Sol and EDL, whereas in Epit muscles both PKC isoforms were reduced by HF diet. In Epit muscles, HF diet also increased the expression of tumor necrosis factor-α (TNF-α) receptors (CD40 and FAS), toll-like receptor 4 (TLR4), and nuclear factor kappa light polypeptide gene enhancer in B cells (NF-kB), whereas in Sol and EDL muscles the expression of these inflammatory genes remained unchanged upon HF feeding. In conclusion, HF diet caused DAG and ceramides accumulation, PKC activation, and the induction of inflammatory pathways in a fiber type-specific manner. These findings help explain why oxidative and glycolytic muscles similarly develop insulin resistance, despite major differences in their metabolic characteristics and responsiveness to dietary lipid abundance.

Exercise maintains euglycemia in association with decreased activation of c-Jun NH2-terminal kinase and serine phosphorylation of IRS-1 in the liver of ZDF rats.

Stress-activated systems and oxidative stress are involved in insulin resistance, which, along with beta-cell failure, contribute to the development of type 2 diabetes mellitus (T2DM). Exercise improves insulin resistance and glucose tolerance, and these adaptations may, in part, be related to reductions in inflammation and oxidative stress. We investigated circulating and tissue-specific markers of inflammation and oxidative stress and insulin-signaling pathways in a rodent model of T2DM, the Zucker diabetic fatty rat, with and without voluntary exercise. At 5 wk of age, Zucker diabetic fatty rats (n = 8-9/group) were divided into basal (B), voluntary exercise (E), and sedentary control (S) groups. B rats were euthanized at 6 wk of age, and S and E rats were euthanized 10 wk later. E rats ran approximately 5 km/day, which improved insulin sensitivity and maintained fed and fasted glucose levels and glucose tolerance. Ten weeks of exercise also decreased whole body markers of inflammation and oxidative stress in plasma and liver, including lowered circulating IL-6, haptoglobin, and malondialdehyde levels, hepatic protein oxidation, and phosphorylated JNK, the latter indicating decreased JNK activity. Hepatic phosphoenolpyruvate carboxykinase levels and Ser(307)-phosphorylated insulin receptor substrate-1 were also reduced in E compared with S rats. In summary, we show that, in a rodent model of T2DM, voluntary exercise decreases circulating markers of inflammation and oxidative stress and lowers hepatic JNK activation and Ser(307)-phosphorylated insulin receptor substrate-1. These changes in oxidative stress markers and inflammation are associated with decreased hyperglycemia and insulin resistance and reduced expression of the main gluconeogenic enzyme phosphoenolpyruvate carboxykinase.

Identification of a WD40 repeat-containing isoform of PHIP as a novel regulator of beta-cell growth and survival.

The pleckstrin homology domain-interacting protein (PHIP) was originally identified as a 902-amino-acid (aa) protein that regulates insulin receptor-stimulated GLUT4 translocation in skeletal-muscle cells. Immunoblotting and immunohistological analyses of pancreatic beta-cells reveal prominent expression of a 206-kDa PHIP isoform restricted to the nucleus. Herein, we report the cloning of this larger, 1,821-aa isoform of PHIP (PHIP1), which represents a novel WD40 repeat-containing protein. We demonstrate that PHIP1 overexpression stimulates insulin-like growth factor 1-dependent and -independent proliferation of beta-cells, an event which correlates with transcriptional upregulation of the cyclin D2 promoter and the accumulation of cyclin D2 protein. RNA interference knockdown of PHIP1 in INS-1 cells abrogates insulin receptor substrate 2 (IRS2)-mediated DNA synthesis, providing for a specific role for PHIP1 in the enhancement of IRS2-dependent signaling responses leading to beta-cell growth. Finally, we provide evidence that PHIP1 overexpression blocks free fatty acid-induced apoptosis in INS-1 cells, which is accompanied by marked activation of phosphoprotein kinase B (PKB)/AKT and the concomitant inhibition of caspase-9 and caspase-3 cleavage. Our finding that the restorative effect of PHIP1 on beta-cell lipotoxicity can be attenuated by the overexpression of dominant-negative PKB suggests a key role for PKB in PHIP1-mediated cytoprotection. Taken together, these findings provide strong support for PHIP1 as a novel positive regulator of beta-cell function. We suggest that PHIP1 may be involved in the induction of long-term gene expression programs to promote beta-cell mitogenesis and survival.

Cold acclimation reduces hepatic protein Kinase B and AMP-activated protein kinase phosphorylation and increases gluconeogenesis in Rats.

This study investigated the molecular and metabolic responses of the liver to cold-induced thermogenesis. To accomplish that, male Wistar rats were exposed to cold (4°C) for 7 days. Livers were then extracted and used for the determination of glucose and fatty acid oxidation, glycogen content, the expression and content of proteins involved in insulin signaling, as well as in the regulation of gluconeogenesis and de novo lipid synthesis. Despite being hyperphagic, cold-acclimated rats displayed normoglycemia with reduced insulinemia, which suggests improved whole-body insulin sensitivity. However, liver protein kinase B (AKT) and glycogen synthase kinase 3 (GSK3) phosphorylations were markedly reduced along with the expressions of the insulin receptor (IR) and its substrates IRS1 and IRS2, whereas glycogen synthase (GS) phosphorylation increased. Thus, major signaling steps of the glycogen synthesis pathway in the liver were inhibited. Furthermore, glucagonemia and hepatic glucose and fatty acid oxidation were increased, whereas liver glycogen content was reduced by cold acclimation. This was accompanied by significantly elevated expressions of the gluconeogenic transcription regulators CRTC2, PGC-1α, and FoxO1, as well as of major gluconeogenic enzymes (G6Pase, FBP1, and PEPCK). Conversely, phosphorylation and contents of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) content were markedly downregulated in livers of cold-acclimated rats. In conclusion, cold acclimation suppressed hepatic glycogen synthesis and promoted profound metabolic changes in the liver so the organ could sustain its ability to regulate whole-body glucose and lipid metabolism under conditions of high-energy demand in thermogenic tissues.

Cold acclimation causes fiber type-specific responses in glucose and fat metabolism in rat skeletal muscles.

This study investigated fiber type-specific metabolic responses and the molecular mechanisms that regulate glucose and fat metabolism in oxidative and glycolytic muscles upon cold acclimation. Male Wistar rats were exposed to cold (4 °C) for 7 days, and then glycogen synthesis and content, glucose and palmitate oxidation, and the molecular mechanisms underlying these metabolic pathways were assessed in soleus (Sol), extensor digitorum longus (EDL), and epitrochlearis (Epit) muscles. Cold acclimation increased glycogen synthesis, glycogen content, glucose oxidation, and reduced glycogen synthase (GS) phosphorylation only in Sol muscles. Protein kinase B (AKT), glycogen synthase kinase 3 (GSK3), and AMP-activated protein kinase (AMPK) phosphorylation increased in all three muscles upon cold acclimation. Cold acclimation increased palmitate oxidation, gene expression of the transcriptional co-activator Pgc-1α, lipoprotein lipase (Lpl), fatty acid transporter (Cd36), and Sarco/endoplasmic reticulum Ca2+-ATPase (Serca) in Sol, EDL, and Epit muscles. Sarcolipin was only detected and had its content increased in Sol muscles. In conclusion, cold-induced thermogenesis activated similar signaling pathways in oxidative and glycolytic muscles, but the metabolic fate of glucose differed in skeletal muscles with distinct fiber type composition. Furthermore, only muscles rich in type I fibers appeared to have the capacity for sarcolipin-mediated SERCA uncoupling.

High-fat diet induces skeletal muscle oxidative stress in a fiber type-dependent manner in rats.

This study investigated the effects of high-fat (HF) diet on parameters of oxidative stress among muscles with distinct fiber type composition and oxidative capacities. To accomplish that, male Wistar rats were fed either a low-fat standard chow (SC) or a HF diet for 8 weeks. Soleus, extensor digitorum longus (EDL), and epitrochlearis muscles were collected and mitochondrial H2O2 (mtH2O2) emission, palmitate oxidation, and gene expression and antioxidant system were measured. Chronic HF feeding enhanced fat oxidation in oxidative and glycolytic muscles. It also caused a significant reduction in mtH2O2 emission in the EDL muscle, although a tendency towards a reduction was also found in the soleus and epitrochlearis muscles. In the epitrochlearis, HF diet increased mRNA expression of the NADPH oxidase complex; however, this muscle also showed an increase in the expression of antioxidant proteins, suggesting a higher capacity to generate and buffer ROS. The soleus muscle, despite being highly oxidative, elicited H2O2 emission rates equivalent to only 20% and 35% of the values obtained for EDL and epitrochlearis muscles, respectively. Furthermore, the Epi muscle with the lowest oxidative capacity was the second highest in H2O2 emission. In conclusion, it appears that intrinsic differences related to the distribution of type I and type II fibers, rather than oxidative capacity, drove the activity of the anti- and pro-oxidant systems and determine ROS production in different skeletal muscles. This also suggests that the impact of potentially deleterious effects of ROS production on skeletal muscle metabolism/function under lipotoxic conditions is fiber type-specific.

Insulin stimulates glucose metabolism via the pentose phosphate pathway in Drosophila Kc cells.

Drosophila melanogaster has become a prominent and convenient model for analysis of insulin action. However, to date very little is known regarding the effect of insulin on glucose uptake and metabolism in Drosophila. Here we show that, in contrast to effects seen in mammals, insulin did not alter [(3)H]2-deoxyglucose uptake and in fact decreased glycogen synthesis ( approximately 30%) in embryonic Drosophila Kc cells. Insulin significantly increased ( approximately 1.5-fold) the production of (14)CO(2) from D-[1-(14)C]glucose while the production of (14)CO(2) from D-[6-(14)C]glucose was not altered. Thus, insulin-stimulated glucose oxidation did not occur via increasing Krebs cycle activity but rather by stimulating the pentose phosphate pathway. Indeed, inhibition of the oxidative pentose phosphate pathway by 6-aminonicotinamide abolished the effect of insulin on (14)CO(2) from D-[U-(14)C]glucose. A corresponding increase in lactate production but no change in incorporation of D-[U-(14)C]glucose into total lipids was observed in response to insulin. Glucose metabolism via the pentose phosphate pathway may provide an important source of 5'-phosphate for DNA synthesis and cell replication. This novel observation correlates well with the fact that control of growth and development is the major role of insulin-like peptides in Drosophila. Thus, although intracellular signaling is well conserved, the metabolic effects of insulin are dramatically different between Drosophila and mammals.

Loss of the anorexic response to systemic 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside administration despite reducing hypothalamic AMP-activated protein kinase phosphorylation in insulin-deficient rats.

This study tested whether chronic systemic administration of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) could attenuate hyperphagia, reduce lean and fat mass losses, and improve whole-body energy homeostasis in insulin-deficient rats. Male Wistar rats were first rendered diabetic through streptozotocin (STZ) administration and then intraperitoneally injected with AICAR for 7 consecutive days. Food and water intake, ambulatory activity, and energy expenditure were assessed at the end of the AICAR-treatment period. Blood was collected for circulating leptin measurement and the hypothalami were extracted for the determination of suppressor of cytokine signaling 3 (SOCS3) content, as well as the content and phosphorylation of AMP-kinase (AMPK), acetyl-CoA carboxylase (ACC), and the signal transducer and activator of transcription 3 (STAT3). Rats were thoroughly dissected for adiposity and lean body mass (LBM) determinations. In non-diabetic rats, despite reducing adiposity, AICAR increased (∼1.7-fold) circulating leptin and reduced hypothalamic SOCS3 content and food intake by 67% and 25%, respectively. The anorexic effect of AICAR was lost in diabetic rats, even though hypothalamic AMPK and ACC phosphorylation markedly decreased in these animals. Importantly, hypothalamic SOCS3 and STAT3 levels remained elevated and reduced, respectively, after treatment of insulin-deficient rats with AICAR. Diabetic rats were lethargic and displayed marked losses of fat and LBM. AICAR treatment increased ambulatory activity and whole-body energy expenditure while also attenuating diabetes-induced fat and LBM losses. In conclusion, AICAR did not reverse hyperphagia, but it promoted anti-catabolic effects on skeletal muscle and fat, enhanced spontaneous physical activity, and improved the ability of rats to cope with the diabetes-induced dysfunctional alterations in glucose metabolism and whole-body energy homeostasis.

Chronic treatment with the AMP-kinase activator AICAR increases glycogen storage and fatty acid oxidation in skeletal muscles but does not reduce hyperglucagonemia and hyperglycemia in insulin deficient rats.

This study tested whether the glycogen-accumulating effect of chronic in vivo pharmacological 5'AMP-activated protein kinase (AMPK) activation could improve glycemic control under conditions of insulin deficiency. Male Wistar rats were rendered diabetic through the administration of streptozotocin (STZ) and then treated for 7 consecutive days with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR). Subsequently, glycogen content and synthesis, glucose oxidation, and fatty acid oxidation (FAO) were determined in oxidative and glycolytic skeletal muscles. Glycemia, insulinemia, glucagonemia, and circulating triglycerides (TG) and non-esterified fatty acids (NEFAs) were measured after AICAR treatment. Insulin was almost undetectable in STZ rats and these animals were severely hyperglycemic. Glycogen content was markedly low mainly in glycolytic muscles of STZ rats and AICAR treatment restored it to control values. No differences were found among all muscles studied with regards to the content and phosphorylation of Akt/protein kinase B and glycogen synthase kinase 3. Even though glycogen synthase content was reduced in all muscles from STZ rats, insulin-induced dephosphorylation/activation of this enzyme was preserved and unaffected by AICAR treatment. Glucagon and NEFAS were 2- and 7.4-fold fold higher in STZ rats than controls, respectively. AICAR did not affect hyperglycemia and hyperglucagonemia in STZ rats; however, it normalized circulating NEFAs and significantly increased FAO in glycolytic muscles. In conclusion, even though AICAR-induced AMPK activation enhanced glycogen accumulation in glycolytic muscles and normalized circulating NEFAs and TG levels, the hyperglycemic effects of glucagon likely offset the potentially glucose-lowering effects of AICAR, resulting in no improvement of glycemic control in insulin-deficient rats.

The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.

The search for putative precursor cells within the pancreas has been the focus of extensive research. Previously, we identified rare pancreas-derived multipotent precursor (PMP) cells in the mouse with the intriguing capacity to generate progeny in the pancreatic and neural lineages. Here, we establish the embryonic pancreas as the developmental source of PMPs through lineage-labeling experiments. We also show that PMPs express insulin and can contribute to multiple pancreatic and neural cell types in vivo. In addition, we have isolated PMPs from adult human islet tissue that are also capable of extensive proliferation, self-renewal, and generation of multiple differentiated pancreatic and neural cell types. Finally, both mouse and human PMP-derived cells ameliorated diabetes in transplanted mice. These findings demonstrate that the adult mammalian pancreas contains a population of insulin(+) multipotent stem cells and suggest that these cells may provide a promising line of investigation toward potential therapeutic benefit.

Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic beta-cells.

Chronic exposure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic beta-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic beta-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced beta-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in beta-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate.

Ex vivo transcriptional profiling of human pancreatic islets following chronic exposure to monounsaturated fatty acids.

The aim of this study was to assess the effects of chronic free fatty acid (FFA) exposure on gene expression and the functional state of human pancreatic islets. Chronic exposure of islets to oleate (OA) resulted in a significant reduction in glucose-stimulated insulin secretion (GSIS) compared with control (466+/-82 vs 234+/-57 ng/microg DNA, P<0.05). OA treatment also led to reduction in total insulin content of the islets (17 609+/-3816 vs 10 599+/-3876 ng insulin/microg DNA) and to an increase in the rate of reactive oxygen species (ROS) generation. Interestingly, the suppressive effects of OA on biosynthesis and secretion of insulin were accompanied by alteration in the expression of 40 genes, as determined by microarray analysis and subsequent qPCR validation. The majority of genes regulated by OA encoded metabolic enzymes. The expression of enzymes involved in oxidative defense was elevated, indicating a link between ROS generation and antioxidant defense activation. Additionally, pretreatment of human islets with OA led to a significant increase (30%) in the rate of oxidation of this fatty acid and to a significant decrease (75%) in glucose oxidation. Importantly, individual analysis of gene clusters from the islets of all donors revealed the induction of genes involved in inflammation and immunity, which provides further evidence that FFA are risk factors for the development of type 2 diabetes. In summary, our data indicate that chronic exposure of human islets to FFA activates inflammatory and metabolic pathways that lead to oxidative stress, reduced beta-cell insulin content, and inhibition of GSIS.

Free fatty acid-induced reduction in glucose-stimulated insulin secretion: evidence for a role of oxidative stress in vitro and in vivo.

OBJECTIVE: An important mechanism in the pathogenesis of type 2 diabetes in obese individuals is elevation of plasma free fatty acids (FFAs), which induce insulin resistance and chronically decrease beta-cell function and mass. Our objective was to investigate the role of oxidative stress in FFA-induced decrease in beta-cell function. RESEARCH DESIGN AND METHODS: We used an in vivo model of 48-h intravenous oleate infusion in Wistar rats followed by hyperglycemic clamps or islet secretion studies ex vivo and in vitro models of 48-h exposure to oleate in islets and MIN6 cells. RESULTS: Forty-eight-hour infusion of oleate decreased the insulin and C-peptide responses to a hyperglycemic clamp (P < 0.01), an effect prevented by coinfusion of the antioxidants N-acetylcysteine (NAC) and taurine. Similar to the findings in vivo, 48-h infusion of oleate decreased glucose-stimulated insulin secretion ex vivo (P < 0.01) and induced oxidative stress (P < 0.001) in isolated islets, effects prevented by coinfusion of the antioxidants NAC, taurine, or tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl). Forty-eight-hour infusion of olive oil induced oxidative stress (P < 0.001) and decreased the insulin response of isolated islets similar to oleate (P < 0.01). Islets exposed to oleate or palmitate and MIN6 cells exposed to oleate showed a decreased insulin response to high glucose and increased levels of oxidative stress (both P < 0.001), effects prevented by taurine. Real-time RT-PCR showed increased mRNA levels of antioxidant genes in MIN6 cells after oleate exposure, an effect partially prevented by taurine. CONCLUSIONS: Our data are the first demonstration that oxidative stress plays a role in the decrease in beta-cell secretory function induced by prolonged exposure to FFAs in vitro and in vivo.

Infection with the cestode Hymenolepis diminuta induces changes in acetylcholine metabolism and muscarinic receptor mRNA expression in the rat jejunum.

Total and neuron-specific uptake of [3H] choline into smooth muscle/myenteric plexus (SM/MP) preparations from the jejunum of rats infected with five Hymenolepis diminuta for 30 days compared to uninfected rats was significantly increased, as was choline acetyltransferase activity and acetylcholine biosynthesis. Although acetylcholinesterase and total cholinesterase activity levels in SM/MP preparations from infected rats were not significantly different from uninfected animals, pseudocholinesterase activity was significantly elevated in infected rats. Infection resulted in a significant elevation in the relative expression of muscarinic 2 (M2) receptor mRNA in jejunum compared to uninfected rats. Conversely, in rats infected with 50 worms for 30 days, the relative expression of muscarinic 1 (M1) receptor mRNA in the jejunum was significantly depressed, while the expression of M2 receptor mRNA was not significantly different from that in five worm infections. The relative expression of muscarinic 3 receptor mRNA was unaffected by infection. The present study shows that infection of rats with low numbers of an enteric cestode leads to a significant modulation of the cholinergic components of the myenteric plexus and M2 receptor mRNA, and that large number of worms result in suppression in the relative expression of M1 receptor mRNA.

The Zn2+-transporting pathways in pancreatic beta-cells: a role for the L-type voltage-gated Ca2+ channel.

In pancreatic beta-cells Zn(2+) is crucial for insulin biosynthesis and exocytosis. Despite this, little is known about mechanisms of Zn(2+) transport into beta-cells or the regulation and compartmentalization of Zn(2+) within this cell type. Evidence suggests that Zn(2+) in part enters neurons and myocytes through specific voltage-gated calcium channels (VGCC). Using a Zn(2+)-selective fluorescent dye with high affinity and quantum yield, FluoZin-3 AM and the plasma membrane potential dye DiBAC(4)(3) we applied fluorescent microscopy techniques for analysis of Zn(2+)-accumulating pathways in mouse islets, dispersed islet cells, and beta-cell lines (MIN6 and beta-TC6f7 cells). Because the stimulation of insulin secretion is associated with cell depolarization, Zn(2+) (5-10 mum) uptake was analyzed under basal (1 mm glucose) and stimulatory (10-20 mm glucose, tolbutamide, tetraethylammonium, and high K(+)) conditions. Under both basal and depolarized states, beta-cells were capable of Zn(2+) uptake, and switching from basal to depolarizing conditions resulted in a marked increase in the rate of Zn(2+) accumulation. Importantly, L-type VGCC (L-VGCC) blockers (verapamil, nitrendipine, and nifedipine) as well as nonspecific inhibitors of Ca(2+) channels, Gd(3+) and La(3+), inhibited Zn(2+) uptake in beta-cells under stimulatory conditions with little or no change in Zn(2+) accumulation under low glucose conditions. To determine the mechanism of VGCC-independent Zn(2+) uptake the expression of a number of ZIP family Zn(2+) transporter mRNAs in islets and beta-cells was investigated. In conclusion, we demonstrate for the first time that, in part, Zn(2+) transport into beta-cells takes place through the L-VGCC. Our investigation demonstrates direct Zn(2+) accumulation in insulin-secreting cells by two pathways and suggests that the rate of Zn(2+) transport across the plasma membrane is dependent upon the metabolic status of the cell.

The characterization of mitochondrial permeability transition in clonal pancreatic beta-cells. Multiple modes and regulation.

Mitochondrial permeability transition (MPT), which contributes substantially to the regulation of normal mitochondrial metabolism, also plays a crucial role in the initiation of cell death. It is known that MPT is regulated in a tissue-specific manner. The importance of MPT in the pancreatic beta-cell is heightened by the fact that mitochondrial bioenergetics serve as the main glucose-sensing regulator and energy source for insulin secretion. In the present study, using MIN6 and INS-1 beta-cells, we revealed that both Ca(2+)-phosphate- and oxidant-induced MPT is remarkably different from other tissues. Ca(2+)-phosphate-induced transition is accompanied by a decline in mitochondrial reactive oxygen species production related to a significant potential dependence of reactive oxygen species formation in beta-cell mitochondria. Hydroperoxides, which are indirect MPT co-inducers active in liver and heart mitochondria, are inefficient in beta-cell mitochondria, due to the low mitochondrial ability to metabolize them. Direct cross-linking of mitochondrial thiols in pancreatic beta-cells induces the opening of a low conductance ion permeability of the mitochondrial membrane instead of the full scale MPT opening typical for liver mitochondria. Low conductance MPT is independent of both endogenous and exogenous Ca(2+), suggesting a novel type of nonclassical MPT in beta-cells. It results in the conversion of electrical transmembrane potential into DeltapH instead of a decrease in total protonmotive force, thus mitochondrial respiration remains in a controlled state. Both Ca(2+)- and oxidant-induced MPTs are phosphate-dependent and, through the "phosphate flush" (associated with stimulation of insulin secretion), are expected to participate in the regulation in beta-cell glucose-sensing and secretory activity.