Increased Plasma Manganese, Partially Reduced Ascorbate,1 and Absence of Mitochondrial Oxidative Stress in Type 2 Diabetes Mellitus: Implications for the Superoxide Uncoupling Protein 2 (Ucp-2) Pathway

Oxidative stress is an important component of diabetes and its complications. Manganese (Mn), the key component of the Mitochondrial antioxidant (MnSOD), plays a key role in the superoxide uncoupling protein 2 (UCP-2) pathway in inhibiting of glucose-stimulated insulin secretion (GSIS). The interactions of Mn with ascorbate and other components of this pathway have not been defined in type-2 diabetes. Fifty established type 2 diabetics (30 males, 20 females) and 30 non-diabetics (controls; 18 males, 12 females) matched for age and sex were investigated. Dietary intake, particularly of micronutrients as assessed by 24-h dietary recall was similar between diabetics and controls. Weight and height of all subjects were determined and body mass index (BMI) computed after clinical assessment. Fasting plasma glucose, manganese, ascorbic acid, creatinine and K⁺ levels were determined; K⁺ was to assess the K⁺ channels, whereas creatinine was to assess probability of oxidative stress nephropathy. Body mass index was greater in DM than in controls (p < 0.001). Fasting plasma glucose and Mn levels (p < 0.00 and p < 0.01, respectively) were higher in diabetes than in the controls. Manganese level was greater than twice the levels in controls. Ascorbic acid was not significantly different (p > 0.05), but was 50% lower than the level in non-diabetics. Potassium like Mn and glucose was significantly higher in diabetes mellitus (DM) than in controls (p < 0.001). Creatinine was not significantly different between diabetics and controls (p > 0.05). Correlations among all parameters were not significantly different. These findings suggest absence of significant oxidative stress in the mitochondria, probably excluding a role for UCP-2-superoxide pathway in the inhibition of glucose-stimulated insulin secretion (GSIS), calling for caution in the precocious conclusion that interruption of UCP-2 activity may provide a viable strategy to improve β-cell dysfunction in type 2 diabetes mellitus.     

ER stress is implicated in mitochondrial dysfunction-induced apoptosis of pancreatic beta cells

Mitochondrial dysfunction induces apoptosis of pancreatic β-cells and leads to type 2 diabetes, but the mechanism involved in this process remains unclear. Chronic endoplasmic reticulum (ER) stress plays a role in the apoptosis of pancreatic β-cells; therefore, in current study, we investigated the implication of ER stress in mitochondrial dysfunction-induced β-cells apoptosis. Metabolic stress induced by antimycin or oligomycin was used to impair mitochondrial function in MIN6N8 cells, which are mouse pancreatic β-cells. Impaired mitochondria dysfunction increased ER stress proteins such as p-eIF2α, GRP78 and GRP 94, as well as ER stress-associated apoptotic factor, CHOP, and activated JNK. AMP-activated protein kinase (AMPK) was also activated under mitochondria dysfunction by metabolic stress. However, the inhibition of AMPK by treatment with compound C, inhibitor of AMPK, and overexpression of mutant dominant negative AMPK (AMPKK45R) blocked the induction of ER stress, which was consist-ent with the decreased β-cell apoptosis and increase of insulin content. Furthermore, mitochondrial dysfunction increased the expression of the inducible nitric oxide synthase (iNOS) gene and the production of nitric oxide (NO), but NO production was prevented by compound C and mutant dominant negative AMPK (AMPK-K45R). Moreover, treatment with 1400W, which is an inhibitor of iNOS, prevented ER stress and apoptosis induced by mitochondrial dysfunction. Treatment of MIN6N8 cells with lipid mixture, physiological conditions of impaired mitochondria function, activated AMPK, increased NO production and induced ER stress. Collectively, these data demonstrate that mitochondrial dysfunction activates AMPK, which induces ER stress via NO production, resulting in pancreatic β-cells apoptosis.     

Oxidative stress in mitochondria

In mitochondria, oxidative phosphorylation and enzymatic oxidation of biogenic amines by monoamine oxidase produce reactive oxygen and nitrogen species, which are proposed to cause neuronal cell death in neurodegenerative disorders, including Parkinson’s and Alzheimer’s disease. In these disorders, mitochondrial dysfunction, increased oxidative stress, and accumulation of oxidation-modified proteins are involved in cell death in definite neurons. The interactions among these factors were studied by use of a peroxynitrite-generating agent, N-morpholino sydnonimine (SIN-1) and an inhibitor of complex I, rotenone, in human dopaminergic SH-SY5Y cells. In control cells, peroxynitrite nitrated proteins, especially the subunits of mitochondrial complex I, as 3-nitrotyrosine, suggesting that neurons are exposed to constant oxidative stress even under physiological conditions. SIN-1 and an inhibitor of proteasome, carbobenzoxy-l-isoleucyl-γ-t-butyl-l-analyl-l-leucinal (PSI), increased markedly the levels of nitrated proteins with concomitant induction of apoptosis in the cells. Rotenone induced mitochondrial dysfunction and accumulation and aggregation of proteins modified with acrolein, an aldehyde product of lipid peroxidation in the cells. At the same time, the activity of the 20S β-subunit of proteasome was reduced significantly, which degrades oxidative-modified protein. The mechanism was proved to be the result of the modification of the 20S β-subunit with acrolein and to the binding of other acrolein-modified proteins to the 20S β-subunit. Increased oxidative stress caused by SIN-1 treatment induced a decline in the mitochondrial membrane potential, ΔΨm, and activated mitochondrial apoptotic signaling and induced cell death in SH-SY5Y cells. As another pathway, p38 mitogen-activated protein (MAP) kinase and exracellular signal-regulated kinase (ERK) mediated apoptosis induced by SIN-1. On the other hand, a series of neuroprotective propargylamine derivatives, including rasagiline [N-propargyl-1(R)aminoindan]and (−)deprenyl, intervened in the activation of apoptotic cascade by reactive oxygen species-reactive nitrogen species in mitochondria through stabilization of the membrane potential, ΔΨm. In addition, rasagiline induced antiapoptotic Bcl-2 and glial cell line-derived neurotrophic factor (GDNF) in SH-SY5Y cells, which was mediated by the ERK-nuclear factor (NF)-κB pathway. These results are discussed in relation to the interaction of oxidative stress and mitochondria in the regulation of neuronal death and survival in neurodegenerative diseases.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Pancreatic β-cell death in response to pro-inflammatory cytokines is distinct from genuine apoptosis

A reduction in functional β-cell mass leads to both major forms of diabetes; pro-inflammatory cytokines, such as interleukin-1beta (IL-1β) and gamma-interferon (γ-IFN), activate signaling pathways that direct pancreatic β-cell death and dysfunction. However, the molecular mechanism of β-cell death in this context is not well understood. In this report, we tested the hypothesis that individual cellular death pathways display characteristic phenotypes that allow them to be distinguished by the precise biochemical and metabolic responses that occur during stimulus-specific initiation. Using 832/13 and INS-1E rat insulinoma cells and isolated rat islets, we provide evidence that apoptosis is unlikely to be the primary pathway underlying β-cell death in response to IL-1β+γ-IFN. This conclusion was reached via the experimental results of several different interdisciplinary strategies, which included: 1) tandem mass spectrometry to delineate the metabolic differences between IL-1β+γ-IFN exposure versus apoptotic induction by camptothecin and 2) pharmacological and molecular interference with either NF-κB activity or apoptosome formation. These approaches provided clear distinctions in cell death pathways initiated by pro-inflammatory cytokines and bona fide inducers of apoptosis. Collectively, the results reported herein demonstrate that pancreatic β-cells undergo apoptosis in response to camptothecin or staurosporine, but not pro-inflammatory cytokines.     

Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids

Western lifestyle plays an important role in the prevalence of type 2 diabetes by causing insulin resistance and pancreatic β-cell dysfunction, a prerequisite for the development of diabetes. High fat diet and alcohol are major components of the western diet. The aim of the present study was to investigate the effects of ethanol and fatty acids on β-cell survival and metabolism. We treated the rat β-cell line RINm5F with ethanol, a mixture of palmitic and oleic acids, or both. Reactive oxygen species (ROS) were determined by (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate) (CM-H2DCFDA) fluorescence assay, and mitochondrial activity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by determining ATP production. Cell viability was assessed with a cell counter and trypan blue exclusion, and the mode of cell death by Hoechst33342 and propidium iodide staining. With both ethanol and fatty acid treatments, MTT reduction and ATP production decreased, whereas ROS production increased. Ethanol treatment had no effect on cell number, whereas fatty acid treatment reduced the cell number. Cell incubation with ethanol, fatty acids, or both increased the number of Hoechst 33342-positive nuclei. However, the majority of nuclei from fatty acid-treated cells were stained with propidium iodide, indicating a loss of plasma membrane integrity. We conclude that both ethanol and fatty acids generate cellular oxidative stress, and affect mitochondrial function in RINm5F β-cells. However, ethanol causes β-cell death by apoptosis, whereas fatty acids cause cell death predominantly by necrosis. It is not known whether these results are applicable to human β-cells.     

Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic β-cells

Advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE) have been linked to the pathogenesis of diabetic complications, such as retinopathy, neuropathy, and nephropathy. AGEs may induce β-cell dysfunction and apoptosis, another complication of diabetes. However, the role of AGE-RAGE interaction in AGE-induced pancreatic β-cell failure has not been fully elucidated. In this study, we investigated whether AGE–RAGE interaction could mediate β-cell failure. We explored the potential mechanisms in insulin secreting (INS-1) cells from a pancreatic β-cell line, as well as primary rat islets. We found that glycated serum (GS) induced apoptosis in pancreatic β-cells in a dose- and time-dependent manner. Treatment with GS increased RAGE protein production in cultured INS-1 cells. GS treatment also decreased bcl-2 gene expression, followed by mitochondrial swelling, increased cytochrome c release, and caspase activation. RAGE antibody and knockdown of RAGE reversed the β-cell apoptosis and bcl-2 expression. Inhibition of RAGE prevented AGE-induced pancreatic β-cell apoptosis, but could not restore the function of glucose stimulated insulin secretion (GSIS) in rat islets. In summary, the results of the present study demonstrate that AGEs are integrally involved in RAGE-mediated apoptosis and impaired GSIS dysfunction in pancreatic β-cells. Inhibition of RAGE can effectively protect β-cells against AGE-induced apoptosis, but cannot reverse islet dysfunction in GSIS.     

Vanadyl bisacetylacetonate protects β cells from palmitate-induced cell death through the unfolded protein response pathway

Endoplasmic reticulum (ER) stress induced by free fatty acids (FFA) is important to β-cell loss during the development of type 2 diabetes. To test whether vanadium compounds could influence ER stress and the responses in their mechanism of antidiabetic effects, we investigated the effects and the mechanism of vanadyl bisacetylacetonate [VO(acac)₂] on β cells upon treatment with palmitate, a typical saturated FFA. The experimental results showed that VO(acac)₂ could enhance FFA-induced signaling pathways of unfolded protein responses by upregulating the prosurvival chaperone immunoglobulin heavy-chain binding protein/78-kDa glucose-regulated protein and downregulating the expression of apoptotic C/EBP homologous protein, and consequently the reduction of insulin synthesis. VO(acac)₂ also ameliorated FFA-disturbed Ca²⁺ homeostasis in β cells. Overall, VO(acac)₂ enhanced stress adaption, thus protecting β cells from palmitate-induced apoptosis. This study provides some new insights into the mechanisms of antidiabetic vanadium compounds.     

In vivo activating transcription factor 3 silencing ameliorates the AMPK compensatory effects for ER stress-mediated β-cell dysfunction during the progression of type-2 diabetes

In obese Zucker diabetic fatty (ZDF) rats, ER stress is associated with insulin resistance and pancreatic β-cell dysfunction; however the exact mechanisms by which ER stress drives type-2 diabetes remain uncertain. Here, we investigated the role of ATF3 on the preventive regulation of AMPK against ER stress-mediated β-cell dysfunction during the end-stage progression of hyperglycemia in ZDF rats. The impaired glucose metabolism and β-cell dysfunction were significantly increased in late-diabetic phase 19-week-old ZDF rats. Although AMPK phosphorylation reduced in 6- and 12-week-old ZDF rats was remarkably increased at 19 weeks, the increases of lipogenice genes, ATF3, and ER stress or ROS-mediated β-cell dysfunction were still remained, which were attenuated by in vivo-injection of chemical chaperon tauroursodeoxycholate (TUDCA), chronic AICAR, or antioxidants. ATF3 did not directly affect AMPK phosphorylation, but counteracts the preventive effects of AMPK for high glucose-induced β-cell dysfunction. Moreover, knockdown of ATF3 by delivery of in vivo-jetPEI ATF3 siRNA attenuated ER stress-mediated β-cell dysfunction and enhanced the beneficial effect of AICAR. Our data suggest that ATF3 may play as a counteracting regulator of AMPK and thus promote β-cell dysfunction and the development of type-2 diabetes and could be a potential therapeutic target in treating type-2 diabetes.     

The critical role of JNK in the ER-mitochondrial crosstalk during apoptotic cell death

Apoptosis or programmed cell death is an extremely coordinated phenomenon that involves the participation of a complex interacting crosstalk between the endoplasmic reticulum and mitochondria. This involves a series of signaling molecules like stress kinases, caspases, Bcl-2 family of proteins, etc. that coordinately induce apoptosis by releasing apoptotic proteins from the mitochondria and mediate DNA damage of the cell. Among the stress kinases, JNK, a member of the MAPK family has been believed to be critically mediating these apoptotic phenomena. The involvement of JNK has been clouded by controversies because of its role both as a pro-apoptotic and an anti-apoptotic mediator. A very significant initiator of JNK activation is the pro-inflammatory cytokine, IL-1β, levels of which are significantly elevated in varied diseases especially diabetes where it is believed to significantly contribute to pancreatic β-cell death. During apoptotic cell death, the endoplasmic reticulum and the mitochondrion participate in a relay of cellular events that determine the onset of the classical apoptotic pathways. Here we discuss the details of this ER-mitochondrial crosstalk and the role of JNK herein that ultimately culminates into apoptotic cell death that is evident in various pathophysiological conditions.     

The cancer cell’s “power plants” as promising therapeutic targets: An overview

This introductory article to the review series entitled “The Cancer Cell’s Power Plants as Promising Therapeutic Targets” is written while more than 20 million people suffer from cancer. It summarizes strategies to destroy or prevent cancers by targeting their energy production factories, i.e., “power plants.” All nucleated animal/human cells have two types of power plants, i.e., systems that make the “high energy” compound ATP from ADP and P _i . One type is “glycolysis,” the other the “mitochondria.” In contrast to most normal cells where the mitochondria are the major ATP producers (>90%) in fueling growth, human cancers detected via Positron Emission Tomography (PET) rely on both types of power plants. In such cancers, glycolysis may contribute nearly half the ATP even in the presence of oxygen (“Warburg effect”). Based solely on cell energetics, this presents a challenge to identify curative agents that destroy only cancer cells as they must destroy both of their power plants causing “necrotic cell death” and leave normal cells alone. One such agent, 3-bromopyruvate (3-BrPA), a lactic acid analog, has been shown to inhibit both glycolytic and mitochondrial ATP production in rapidly growing cancers (Ko et al., Cancer Letts., 173, 83–91, 2001), leave normal cells alone, and eradicate advanced cancers (19 of 19) in a rodent model (Ko et al., Biochem. Biophys. Res. Commun., 324, 269–275, 2004). A second approach is to induce only cancer cells to undergo “apoptotic cell death.” Here, mitochondria release cell death inducing factors (e.g., cytochrome c). In a third approach, cancer cells are induced to die by both apoptotic and necrotic events. In summary, much effort is being focused on identifying agents that induce “necrotic,” “apoptotic” or apoptotic plus necrotic cell death only in cancer cells. Regardless how death is inflicted, every cancer cell must die, be it fast or slow.     

Sweet Killing in Obesity and Diabetes: The Metabolic Role of the BH3-only Protein BIM

Diabetes is a metabolic disorder affecting more than 400 million individuals and their families worldwide. The major forms of diabetes (types 1 and 2) are characterized by pancreatic β-cell dysfunction and, in some cases, loss of β-cell mass causing hyperglycemia due to absolute or relative insulin deficiency. The BCL-2 homology 3 (BH3)-only protein BIM has a wide role in apoptosis induction in cells. In this review, we describe the apoptotic mechanisms mediated by BIM activation in β cells in obesity and both forms of diabetes. We focus on molecular pathways triggered by inflammation, saturated fats, and high levels of glucose. Besides its role in cell death, BIM has been implicated in the regulation of mitochondrial oxidative phosphorylation and cellular metabolism in hepatocytes. BIM is both a key mediator of pancreatic β-cell death and hepatic insulin resistance and is thus a potential therapeutic target for novel anti-diabetogenic drugs. We consider the implications and challenges of targeting BIM in the treatment of the disease.     

Stem cell potential for type 1 diabetes therapy

Stem cells have been considered as a useful tool in Regenerative Medicine due to two main properties: high rate of self-renewal, and their potential to differentiate into all cell types present in the adult organism. Depending on their origin, these cells can be grouped into embryonic or adult stem cells. Embryonic stem cells are obtained from the inner cell mass of blastocyst, which appears during embryonic day 6 of human development. Adult stem cells are present within various tissues of the organism and are responsible for their turnover and repair. In this sense, these cells open new therapeutic possibilities to treat degenerative diseases such as type 1 diabetes. This pathology is caused by the autoimmune destruction of pancreatic β-cells, resulting in the lack of insulin production. Insulin injection, however, cannot mimic β-cell function, thus causing the development of important complications. The possibility of obtaining β-cell surrogates from either embryonic or adult stem cells to restore insulin secretion will be discussed in this review.     

Inhibitors of islet amyloid polypeptide fibrillogenesis, and the treatment of type-2 diabetes

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. Although it may be a secondary event in the etiology of diabetes, the accumulation of insoluble IAPP fibrils is considered to be a primary cause of β-cell failure in affected individuals. A possible means of inhibiting this process is through the use of small peptides that bind to IAPP and prevent fibril polymerization. This approach has been examined using a series of overlapping hexamers that target the known amyloidogenic regions of IAPP. Peptides were examined usingin vitroassays and active inhibitors were identified by their ability to prevent amyloid-related conformational transitions and IAPP aggregation. Fragments such as those corresponding to the IAPP-derived sequences, SNNFGA (residues 20–25) and GAILSS (residues 24–29), were potent inhibitors ofβ-sheet folding and amyloid fibril formation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP significantly decreased the density of fibrils and any remaining structures displayed altered morphology. In some, but not all cases, inhibition of amyloid fibrils also correlated with an ability to reduce IAPP-mediated cytotoxicity as determined in cell culture studies. The results from these studies suggest that these two peptide inhibitors differ in their mechanisms of action possibly due to unique interactions with the full-length IAPP molecule. These inhibitors form the basis of a therapeutic strategy to prevent amyloid accumulation leading to improved islet survival and a potentially novel treatment for type-2 diabetes.     

Upregulation of PTEN by peroxynitrite contributes to cytokine-induced apoptosis in pancreatic β-cells

Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, by negatively regulating the PI3K-Akt signaling pathway, participates in multiple biological processes such as cell proliferation, apoptosis, differentiation, and migration. Recent studies show that selective deletion of PTEN in pancreatic β-cells leads to resistance to streptozotocin (STZ)-induced diabetes, but the mechanism is unclear. One major mechanism underlying STZ toxicity is cytokine-mediated β-cell destruction in which oxidative stress plays a key role. The present study investigated the role of PTEN in cytokine-induced β-cell apoptosis, and further explored whether oxidative stress, particularly peroxynitrite formation, could regulate PTEN-Akt pathway. Incubation of βTC-6 cells with cytokine mixture (IL-1β, TNF-α, and IFN-γ) or exogenous peroxynitrite significantly increased apoptotic cell percentage, elevated PTEN and p-PTEN levels, and inhibited Akt activation. Transfection with PTEN-specific siRNA protected βTC-6 cells from cytokine or peroxynitrite-mediated cell apoptosis and partially reversed Akt inhibition. Furthermore, nitrotyrosine formation, an indicator of peroxynitrite production, was significantly elevated after cytokine treatment. Preventing peroxynitrite formation by administrating NAC/l-NMMA, or scavenging peroxynitrite directly by UA, attenuated cytokine-induced PTEN upregulation, Akt inhibition, and β-cell apoptosis. These findings suggest that peroxynitrite-mediated PTEN upregulation plays an important role in cytokine-induced pancreatic β-cell apoptosis.     

Role of the mitochondrial membrane permeability transition in cell death

In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic or necrotic death, although the details of the mechanism involved remain to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca²⁺-dependent increase of mitochondrial membrane permeability that leads to loss of Δψ, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel that is known as the permeability transition pore (PTP), which putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Recently, significant progress has been made by studies performed with mice lacking Cyp D at several laboratories, which have convincingly demonstrated that Cyp D is essential for the MPT to occur and that the Cyp D-dependent MPT regulates some forms of necrotic, but not apoptotic, cell death. Cyp D-deficient mice have also been used to show that the Cyp D-dependent MPT plays a crucial role in ischemia/reperfusion injury. The anti-apoptotic proteins Bcl-2 and Bcl-x_L have the ability to block the MPT, and can therefore block MPT-dependent necrosis in addition to their well-established ability to inhibit apoptosis.     

Glucagon-Like Peptide-1 Induced Signaling and Insulin Secretion Do Not Drive Fuel and Energy Metabolism in Primary Rodent Pancreatic β-Cells

Background

Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic β-cells, in particular cAMP, Ca²⁺ and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors.

Methodology/Prinicipal Findings

GLP-1 or Ex-4 at high glucose caused release (∼20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on β-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca²⁺]_i and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered.

Conclusions/Significance

The results indicate that GLP-1 barely affects β-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the β-cell, and that the β-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a “push” (fuel substrate driven) process, rather than a “pull” mechanism secondary to enhanced insulin release as well as to Ca²⁺, cAMP and PKB signaling.     

Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells

The mechanisms related to hyperglycemia-induced pancreatic β-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Δ < eqid1 > _m), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Δ < eqid2 > _m, lead to an important pancreatic β-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia. This work is part of the thesis required for the doctorate degree in Biological Sciences at the Universidad Autónoma Metropolitana, Mexico City, Mexico.     

The Role of TNF-<Emphasis Type="Italic">α</Emphasis> and its Receptors in the Production of <Emphasis Type="Italic">β</Emphasis>-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

β-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation. Chunlin Xia is the co-first author.