No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Prohibitin is expressed in pancreatic β-cells and protects against oxidative and proapoptotic effects of ethanol
The FEBS Journal
Abstract
Pancreatic beta-cell dysfunction is a prerequisite for the development of type 2 diabetes. Alcoholism is a diabetes risk factor and ethanol increases oxidative stress in beta-cells, whereas the mitochondrial chaperone prohibitin (PHB) has antioxidant effects in several cell types. In the present study we investigated whether PHB is expressed in beta-cells and protects these cells against deleterious effects of ethanol, using INS-1E and RINm5F beta-cell lines. Endogenous PHB was detected by western blot and immunocytochemistry. Reactive oxygen species were determined by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate fluorescence assay, and mitochondrial activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, uncoupling protein 2 expression and ATP production. Cell death was determined by Hoechst 33342 staining, cleaved caspase-3 levels and flow cytometry. PHB was expressed in beta-cells under normal conditions and colocalized with Hoechst 33342 in the nucleus and with the mitochondrial probe Mitofluor in the perinuclear area. In ethanol-treated cells, MTT reduction and ATP production decreased, whereas reactive oxygen species, uncoupling protein 2 and cleaved caspase-3 levels increased. In addition, flow cytometry analysis showed an increase of apoptotic cells. Ethanol treatment increased PHB expression and induced PHB translocation from the nucleus to the mitochondria. PHB overexpression decreased the apoptotic effects of ethanol, whereas PHB knockdown enhanced these effects. The protective effects of endogenous PHB were recapitulated by incubation of the cells with recombinant human PHB. Thus, PHB is expressed in beta-cells, increases with oxidative stress and protects the cells against deleterious effects of ethanol.
... [6][7][8][9][10][11] Unfortunately, although they have provided mechanistic insight, these studies have not translated into new therapies to combat organ dysfunction in sepsis, and mortality for patients in septic shock remains as high today (≈30%) as it was several decades ago. 12,13 PHB1 and PHB2 (prohibitins) are proteins that assemble in hetero-oligomeric complexes within the mitochondrial inner membrane and in plasma membrane lipid rafts where they play critical roles in mitochondrial function and metabolism, [14][15][16][17][18] cellular proliferation and tumorigenesis, [19][20][21] inflammation/oxidative stress, 22 and apoptosis. [23][24][25] Little information exists regarding the function of PHBs in the heart, although overexpression of PHB1 was shown to protect against oxidative insults in cardiomyocytes. ...
... Previous studies have reported that PHB1 protects cells against various stressors, including oxidative stress and inflammation. 22,28,42 To examine whether PHB1 is acting in similar manner during acute inflammation, HL-1 cardiomyocytes were transfected with plasmid encoding PHB1 with a GFP (green fluorescent protein) tag under control of CMV promoter (overexpression of PHB1), or GFP vector alone (Vec). As expected, localization of PHB1-GFP in normal, unstressed HL-1 cardiomyocytes was primarily mitochondrial ( Figure 2A). ...
... Overexpression of PHB1 was shown in a prior report to protect cardiomyocytes against oxidative stress in prior studies, although the mechanisms of protection were not clearly determined. 26,27 Interestingly, recombinant human PHB1 (rPHB1) protected pancreatic cells against oxidative stress induced by ethanol, 22 suggesting that PHB1 also acts via exogenous mechanisms. To gain mechanistic insight into these protective effects of PHB1, HL-1 cardiomyocytes were transfected with a lentiviral-based reporter system containing tandem repeats of the antioxidant response element promoter controlling expression of GFP. ...
Background
In sepsis, circulating cytokines and lipopolysaccharide elicit mitochondrial dysfunction and cardiomyopathy, a major cause of morbidity and mortality with this condition. Emerging research places the PHB1 (lipid raft protein prohibitin‐1) at the nexus of inflammation, metabolism, and oxidative stress. PHB1 has also been reported in circulation, though its function in this compartment is completely unknown.
Methods and Results
Using a wide‐ranging approach across multiple in vitro and in vivo models, we interrogated the functional role of intracellular and circulating PHB1 in the heart during sepsis, and elucidated some of the mechanisms involved. Upon endotoxin challenge or sepsis induction in rodent models, PHB1 translocates from mitochondria to nucleus in cardiomyocytes and is secreted into the circulation from the liver in a manner dependent on nuclear factor (erythroid‐derived 2)‐like 2, a key transcriptional regulator of the antioxidant response. Overexpression or treatment with recombinant human PHB1 enhances the antioxidant/anti‐inflammatory response and protects HL‐1 cardiomyocytes from mitochondrial dysfunction and toxicity from cytokine stress. Importantly, administration of recombinant human PHB1 blunted inflammation and restored cardiac contractility and ATP production in mice following lipopolysaccharide challenge. This cardioprotective, anti‐inflammatory effect of recombinant human PHB1 was determined to be independent of nuclear factor (erythroid‐derived 2)‐like 2, but partially dependent on PI3K/AKT signaling in the heart.
Conclusions
These findings reveal a previously unknown cardioprotective effect of PHB1 during sepsis, and illustrate a pro‐survival, protective role for PHB1 in the circulation. Exploitation of circulating PHB1 as a biomarker and/or therapeutic could have widespread benefit in the clinical management of sepsis and other severe inflammatory disorders.
... 5 There are three major domains in the protein sequence of PHB1: the N-terminal hydrophobic alpha helix that functions as a membrane-anchoring domain, the mid-region that contains the PHB domain, and the C-terminus that contains a coiled-coil domain with a small nuclear localization sequence. 6 PHB1 has been implicated in diverse fundamental cellular functions, such as cell proliferation, 7 cell cycle control, 8 differentiation 9 and protection from oxidative stress, 10 and it acts as a molecular chaperone. 11 PHB1 is a ubiquitously expressed protein that is present in various cellular compartments, such as the cell membrane, 12 mitochondria 7,13 and nucleus. ...
... There are several reports in the literature showing PHB1 is translocated from mitochondria to the plasma membrane, 18,37 and from the nucleus to mitochondria upon activation by various stimuli or agents. 10,38,39 However, the mechanisms involved in the intracellular trafficking of PHB1 are not well understood. We have shown that PHB1 undergoes palmitoylation at Cys-69 and interacts with Eps 15 homology domain protein 2 (EHD2). ...
... 62 There are numerous reports in the literature that provide support for PHB1 having a protective role in oxidative stress and mitochondrial dysfunction. 10,63,64 It is possible that the iron-binding function of PHB1 plays a role in its protective effects against oxidative stress. We propose that the Tyr-114 residue in PHB1 has functions that are unique to each cellular compartment. ...
Prohibitin is a pleiotropic protein that has roles in fundamental cellular processes, such as cellular proliferation and mitochondrial housekeeping, and in cell- or tissue-specific functions, such as adipogenesis and immune cell functions. The different functions of prohibitin are mediated by its cell compartment-specific attributes, which include acting as an adaptor molecule in membrane signaling, a scaffolding protein in mitochondria, and a transcriptional co-regulator in the nucleus. However, the precise relationship between its distinct cellular localization and diverse functions remain largely unknown. Accumulating evidence suggests that the phosphorylation of prohibitin plays a role in a number of cell signaling pathways and in intracellular trafficking. Herein, we discuss the known and potential importance of the site-specific phosphorylation of prohibitin in regulating these features. We will discuss this in the context of new evidence from tissue-specific transgenic mouse models of prohibitin, including a mutant prohibitin lacking a crucial tyrosine phosphorylation site. We conclude with the opinion that prohibitin can be used as a potential target for tyrosine kinase signal transduction-targeting therapy, including in insulin, growth factors, and immune signaling pathways.
... PHB expression is enhanced in neurons by various stressors, including electrical stimulation, hypoxia ischemia, oxygen-glucose deprivation (Zhou et al., 2012), exercise-induced neuroplasticity (Ding et al., 2006), injection of the neurotoxin 6hydroxydopamine (Park et al., 2010), and schizophrenia-induced oligodendrocyte dysfunction (Bernstein et al., 2012). PHB is also up-regulated in the liver of steatohepatitis patients (Tsutsumi et al., 2009), in fetal rabbit lung after exposure to hyperoxic conditions (Henschke et al., 2006), in pancreatic β-cells after ethanol intoxication (Lee et al., 2010a), and in cardiac cells after ischemic-hypoxic preconditioning (Kim et al., 2006;Muraguchi et al., 2010) or chronic restraint stress (Liu et al., 2004). This cytoprotective response involves the translocation of PHB from the nucleus and the cytoplasm to mitochondria in pancreatic β-cells (Lee et al., 2010a), in the retina and retinal epithelium (Lee et al., 2010a), and in ovarian GCs (Chowdhury et al., 2007(Chowdhury et al., , 2011(Chowdhury et al., , 2013Wang et al., 2013). ...
... PHB is also up-regulated in the liver of steatohepatitis patients (Tsutsumi et al., 2009), in fetal rabbit lung after exposure to hyperoxic conditions (Henschke et al., 2006), in pancreatic β-cells after ethanol intoxication (Lee et al., 2010a), and in cardiac cells after ischemic-hypoxic preconditioning (Kim et al., 2006;Muraguchi et al., 2010) or chronic restraint stress (Liu et al., 2004). This cytoprotective response involves the translocation of PHB from the nucleus and the cytoplasm to mitochondria in pancreatic β-cells (Lee et al., 2010a), in the retina and retinal epithelium (Lee et al., 2010a), and in ovarian GCs (Chowdhury et al., 2007(Chowdhury et al., , 2011(Chowdhury et al., , 2013Wang et al., 2013). Sripathi et al. (2011) suggested that the localization and trafficking of PHBs are determined by the modulation of their binding activities to specific lipids. ...
... PHB is also up-regulated in the liver of steatohepatitis patients (Tsutsumi et al., 2009), in fetal rabbit lung after exposure to hyperoxic conditions (Henschke et al., 2006), in pancreatic β-cells after ethanol intoxication (Lee et al., 2010a), and in cardiac cells after ischemic-hypoxic preconditioning (Kim et al., 2006;Muraguchi et al., 2010) or chronic restraint stress (Liu et al., 2004). This cytoprotective response involves the translocation of PHB from the nucleus and the cytoplasm to mitochondria in pancreatic β-cells (Lee et al., 2010a), in the retina and retinal epithelium (Lee et al., 2010a), and in ovarian GCs (Chowdhury et al., 2007(Chowdhury et al., , 2011(Chowdhury et al., , 2013Wang et al., 2013). Sripathi et al. (2011) suggested that the localization and trafficking of PHBs are determined by the modulation of their binding activities to specific lipids. ...
Prohibitins are members of a highly conserved protein family containing the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain [also known as the prohibitin (PHB) domain] found unicellular eukaryotes, fungi, plants, animals and humans. Two highly homologous members of prohibitins expressed in eukaryotes are prohibitin (PHB; B-cell receptor associated protein-32, BAP-32) and prohibitin 2/repressor of estrogen receptor activity (PHB2, REA, BAP-37). Both PHB and REA/PHB2 are ubiquitously expressed and are present in multiple cellular compartments including the mitochondria, nucleus, and the plasma membrane. Multiple functions have been attributed to the mitochondrial and nuclear PHB and PHB2/REA including cellular differentiation, anti-proliferation, and morphogenesis. One of the major functions of the prohibitins are in maintaining the functional integrity of the mitochondria and protecting cells from various stresses. In the present review, we focus on the recent research developments indicating that PHB and PHB2/REA is involved in maintaining cellular survival through the Ras-Raf-MEK-Erk pathway. Understanding the molecular mechanisms by which the intracellular signaling pathways utilize prohibitins in governing cellular survival is likely to result in development of therapeutic strategies to overcome various human pathological disorders such as diabetes, obesity, neurological diseases, inflammatory bowel disease and cancer. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
... Prolonged alcohol intake at high doses induces cell damage. Alcohol cytotoxicity is well studied in a variety of tissues including liver, brain, kidney, and gastrointestinal tract [5][6][7][8][9][10][11]. These studies have indicated that ethanol induces apoptosis through oxidative stress, mitochondrial dysfunction, and caspase activation [9][10][11][12][13][14]. ...
... In rats, chronic treatment of ethanol reduced the volume of insulin-secreting βcells [40] and basal and glucose-stimulated insulin secretion [41]. In addition, ethanol induces β-cell apoptosis via mitochondrial dysfunction and oxidative stress [8,42]. For pancreatic ductal epithelial cells, modulation of secretory functions by ethanol has been reported. ...
... In agreement with previous reports [8,12,14], depolarized MMP in PDEC leads to the activation of caspase-3, an initial caspase in apoptosis. It has been shown in several studies that caspase-3 is activated by cytochrome C release from the mitochondria due to MMP depolarization [1,2,59]. ...
Alcohol abuse is a major cause of pancreatitis. However alcohol toxicity has not been fully elucidated in the pancreas and little is known about the effect of alcohol on pancreatic ducts. We report the molecular mechanisms of ethanol-induced damage of pancreatic duct epithelial cells (PDEC). Ethanol treatment for 1, 4, and 24 h resulted in cell death in a dose-dependent manner. The ethanol-induced cell damage was mainly apoptosis due to generation of reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (MMP), and activation of caspase-3 enzyme. The antioxidant N-acetylcysteine (NAC) attenuated these cellular responses and reduced cell death significantly, suggesting a critical role for ROS. Acetaldehyde, a metabolic product of alcohol dehydrogenase, induced significant cell death, depolarization of MMP, and caspase-3 activation as ethanol and this damage was also averted by NAC. Reverse transcription-polymerase chain reaction revealed the expression of several subtypes of alcohol dehydrogenase and acetaldehyde dehydrogenase. Nuclear magnetic resonance spectroscopy data confirmed the accumulation of acetaldehyde in ethanol-treated cells, suggesting that acetaldehyde formation can contribute to alcohol toxicity in PDEC. Finally, ethanol increased the leakage of PDEC monolayer which was again attenuated by NAC. In conclusion, ethanol induces apoptosis of PDEC and thereby may contribute to the development of alcohol-induced pancreatitis.
... Indeed, PHB1 expression is enhanced in neurons by various stresses, including electrical stimulation, hypoxia-ischemia, oxygen-glucose deprivation (Zhou et al., 2012), exercise-induced neuroplasticity (Ding et al., 2006), injection of the neurotoxin 6-hydroxydopamine (Park et al., 2010), and schizophrenia-induced oligodendrocyte dysfunction (Bernstein et al., 2012). PHB1 is also upregulated in the liver of steatohepatitis patients (Tsutsumi et al., 2009), in fetal rabbit lung after exposure to hyperoxic conditions (Henschke et al., 2006), in pancreatic b-cells after ethanol intoxication, (Lee et al., 2010b), and in cardiac cells after ischemichypoxic preconditioning Muraguchi et al., 2010) or chronic restraint stress . This cytoprotectant response involves the translocation of PHB1 from the nucleus and the cytoplasm to mitochondria in pancreatic b-cells (Lee et al., 2010b), in the retina and retinal epithelium (Lee et al., 2010a), and in ovarian granulosa cells (Chowdhury et al., 2007; Figure S2). ...
... PHB1 is also upregulated in the liver of steatohepatitis patients (Tsutsumi et al., 2009), in fetal rabbit lung after exposure to hyperoxic conditions (Henschke et al., 2006), in pancreatic b-cells after ethanol intoxication, (Lee et al., 2010b), and in cardiac cells after ischemichypoxic preconditioning Muraguchi et al., 2010) or chronic restraint stress . This cytoprotectant response involves the translocation of PHB1 from the nucleus and the cytoplasm to mitochondria in pancreatic b-cells (Lee et al., 2010b), in the retina and retinal epithelium (Lee et al., 2010a), and in ovarian granulosa cells (Chowdhury et al., 2007; Figure S2). Sripathi et al. (2011) suggested that the localization and trafficking of PHBs are determined by the modulation of their binding to specific lipids. ...
... Overexpression of PHB1 protects pancreatic b-cells, ovarian granulosa cells, and cardiomyocytes from apoptosis induced by ethanol, ceramide, staurosporine, serum withdrawal, and oxidative stress-induced injury, consistent with PHB1 having a cytoprotective role (Chowdhury et al., 2007Lee et al., 2010b;Liu et al., 2009b). Merkwirth et al. (2012) recently described a critical role of PHB2 in the survival of neurons. ...
Prohibitins (PHBs) are scaffold proteins that modulate many signaling pathways controlling cell survival, metabolism, and inflammation. Several drugs that target PHBs have been identified and evaluated for various clinical applications. Preclinical and clinical studies indicate that these PHB ligands may be useful in oncology, cardiology, and neurology, as well as against obesity. This review covers the physiological role of PHBs in health and diseases and current developments concerning PHB ligands.
... We have been interested in understanding the roles and regulation of PHB1, including its cell type-specific functions, such as adipocytes and pancreatic beta cells 10,11 . Recently, using a transgenic mouse model of PHB1 (Fabp4-PHB1-Tg), we have shown that PHB1 plays a crucial role in lipid homeostasis, mediated through its mitochondrial functions 12 . ...
... The findings, along with current knowledge in literature 11,12,30 , indicate that the mitochondrial attributes of PHB1 contribute to the cell type-specific functions of PHB1 in lipid homeostasis in adipocytes 12,30 and steroidogenic cells 13 , which further supports our conclusion that PHB1 plays a multifaceted role in adrenocortical cells. Altered cholesterol homeostasis and reduced glucocorticoid production from adrenocortical cells in response to PHB1 knockdown strengthens the role of PHB1 in corticosteroidogenesis. ...
Cell-intrinsic early events involved in different trophic hormone-induced steroidogenesis in their respective steroidogenic cell type are very similar. For example, the activation of the cAMP-PKA signaling pathway in response to trophic hormone stimulation and, subsequently, cholesterol transport to the mitochondria to initiate steroidogenesis is common to them. Recently, we have found that an evolutionarily conserved protein, prohibitin-1 (PHB1), is regulated by Luteinizing Hormone (LH) in murine Leydig cells and plays a role in interconnected cell signaling and mitochondrial steps pertaining to testosterone production. Among the primary steroidogenic tissues, PHB1 expression levels are highest in the adrenal cortex (The Human Protein Atlas); however, its regulation and role in adrenocortical cells are virtually unknown. We investigated the regulation and the role of PHB1 in adrenocortical cells in vitro using human HAC15 and mouse Y-1 cell culture models. It was found that Adrenocorticotrophic Hormone (ACTH) stimulation upregulates PHB1 levels in adrenocortical cells in a time-dependent manner. A similar effect on PHB1 levels was also observed in response to dibutyryl-cAMP stimulation, a cell-permeable analogue of cAMP (the second messenger for ACTH action). Moreover, manipulating PHB1 levels in adrenocortical cells affected mitochondria, lysosomes, and lipid droplet characteristics, modulated phospho-PKA and phospho-ERK1/2 levels, and altered corticosteroid production. This finding suggests that ACTH regulates PHB1 in adrenocortical cells and plays a role in corticosteroid production, which was previously unknown.
... exposure-induced pancreatic β-cell dysfunction and apoptosis. 6,[9][10][11] Thus, antioxidant agents may be helpful in the reduction of β-cells damage induced by ethanol, thereby preventing T2DM development stimulated by chronic alcohol ingestion. ...
... It is well established that ethanol-induced β-cell destruction is associated with mitochondrial dysfunction, oxidative stress and increased production of ROS in pancreatic islets. 6,7,35,36 Consistent with previous studies, we observed ROS accumulation, elevated MDA and 4-HNE (markers of oxidative stress) in pancreas and islets for EtOH-fed mice, while LNT administration dramatically reversed those effects. Historically, pancreatic β-cells are considered to be highly susceptible to oxidative stress since they express very low levels of antioxidant enzymes including catalase (Cat), glutathione peroxidase (Gpx) and superoxide dismutase (Sod). ...
Chronic ethanol consumption is a well‐established independent risk factor for type 2 diabetes mellitus (T2DM). Recently, increasing studies have confirmed that excessive heavy ethanol exerts direct harmful effect on pancreatic β‐cell mass and function, which may be a mechanism of pancreatic β‐cell failure in T2DM. In this study, we evaluated the effect of Lentinan (LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β‐cell apoptosis and dysfunction caused by ethanol and the possible mechanisms implicated. Functional studies reveal that LNT attenuates chronic ethanol consumption‐induced impaired glucose metabolism in vivo. In addition, LNT ameliorates chronic ethanol consumption‐induced β‐cell dysfunction, which is characterized by reduced insulin synthesis, defected insulin secretion and increased cell apoptosis. Furthermore, mechanistic assays suggest that LNT enhances β‐cell antioxidant capacity and ameliorates ethanol‐induced oxidative stress by activating Nrf‐2 antioxidant pathway. Our results demonstrated that LNT prevents ethanol‐induced pancreatic β‐cell dysfunction and apoptosis, and therefore may be a potential pharmacological agent for preventing pancreatic β‐cell failure associated with T2DM and stress‐induced diabetes.
... PHB is involved cell signaling, aging, regulation of transcription and acts a chaperone for imported proteins in the mitochondria [46][47][48][49]. Several studies have shown that over-expression of prohibitin protects cells from oxidative stress injury and its expression in cultured cardiomyocytes and in pancreatic β-cells [46,50]. ...
... PHB is involved cell signaling, aging, regulation of transcription and acts a chaperone for imported proteins in the mitochondria [46][47][48][49]. Several studies have shown that over-expression of prohibitin protects cells from oxidative stress injury and its expression in cultured cardiomyocytes and in pancreatic β-cells [46,50]. PHB expression stabilizes the mitochondrial oxidative phosphorylation system in the inner mitochondrial membrane [47]. ...
Background:
Excessive reactive oxygen species from endothelial mitochondria in type 2 diabetes individuals (T2DM) may occur through multiple related mechanisms, including production of mitochondrial reactive oxygen species (mtROS), inner mitochondrial membrane (Δψm) hyperpolarization, changes in mitochondrial mass and membrane composition, and fission of the mitochondrial networks. Inner mitochondrial membrane proteins uncoupling protein-2 (UCP2) and prohibitin (PHB) can favorably impact mtROS and mitochondrial membrane potential (Δψm). Circulating levels of UCP2 and PHB could potentially serve as biomarker surrogates for vascular health in patients with and without T2DM.
Methods:
Plasma samples and data from a total of 107 individuals with (N = 52) and without T2DM (N = 55) were included in this study. Brachial artery flow mediated dilation (FMD) was measured by ultrasound. ELISA was performed to measure serum concentrations of PHB1 and UCP2. Mitochondrial membrane potential was measured from isolated leukocytes using JC-1 dye.
Results:
Serum UCP2 levels were significantly lower in T2DM subjects compared to control subjects (3.01 ± 0.34 vs. 4.11 ± 0.41 ng/mL, P = 0.04). There were no significant differences in levels of serum PHB. UCP2 levels significantly and positively correlated with FMDmm (r = 0.30, P = 0.03) in T2DM subjects only and remained significant after multivariable adjustment. Within T2DM subjects, serum PHB levels were significantly and negatively correlated with UCP2 levels (ρ = - 0.35, P = 0.03).
Conclusion:
Circulating UCP2 levels are lower in T2DM patients and correlate with endothelium-dependent vasodilation in conduit vessels. UCP2 could be biomarker surrogate for overall vascular health in patients with T2DM and merits additional investigation.
... Accumulation of Phb in mitochondria may also be accounted for by other reasons. In the cells of animals, it was shown that prohibitins may travel from the mitochondria to the nucleus [8] and back under stress conditions [9,29]. Therefore, a lack of balance between a rise in Phb3 content in total cellular protein and in the mitochondria may be accounted for by the fact that heat treatment causes not only changes in the content of prohibitins but also results in their redistribution within the cell. ...
... It is possible that total content of prohibitins changes moderately, but their accumulation in mitochondria is appreciable. However, one should remember that association of prohibitins with the nuclear membrane was shown only in the cells of animals [8,29]. ...
In the organelles of animals, plants, and microorganisms, highly conserved membrane proteins prohibitins (Phb) govern some of the protein–membrane interactions. Changes in expression of genes phb3 and phb4 in arabidopsis induce the genes that are also activated by oxidative, salt, and other abiotic stresses. Prohibitins are assumed to participate in the modulation of the state of mitochondrial membranes that are an important element of stress response. Thus, prohibitins may influence the development of response to stress. However, the data directly indicating a relationship between plant prohibitins and adaptation to stress are not available. It was shown that preliminary hardening of arabidopsis plants at 45°C protected leaves under a severe heat shock (50°C). After exposure to different hardening temperatures, the content of proteins Phb3 and Phb4 in the leaves of arabidopsis reliably increased several hours after the termination of stress, and we observed a considerable rise in the content of phb3 and phb4 transcripts and accumulation of proteins Phb3 and Phb4 in the mitochondria right after the treatment at a temperature of 45°C. Possible reasons for this accumulation are discussed.
... In addition to the exocrine (acinar cells) pancreas, endocrine pancreas (islet cells) is also affected by fibrosis. Despite this, diabetes only becomes evident at the later stages of the disease [6]. ...
... In the present study, group II showed a marked damage and distortion of the pancreatic acini. These findings were in agreement with Lee et al. [6] who related this acinar damage to the oxidative stress induced by ethanol. They added that ethanol is metabolized by three different pathways: alcohol dehydrogenase, microsomal ethanol oxidation system, and catalase. ...
... Pancreatic β-cell dysfunction is a prerequisite for the development of type 2 diabetes Alcoholism is a risk factor and ethanol increases oxidative stress in beta-cells. Cells in a such state increase expression of PHB (poly3-hydroxybutyric acid) synthase genes, to protect them from the harmful effects of ethanol [3]. ...
... Stimulation of insulin secretion with diacylglycerol is explained by the release of Ca from endoplasmic reticulum [20]. Response to high glucose stimulation decreases due to reduced levels of cAMP caused by reduced levels of ATP [2,3,21]. ...
Introduction/Objective. There are controversial data in the literature on the topic of effects of alcohol on insulin secretion, apoptosis, and necrosis of the endocrine and exocrine pancreas. The goal of this research was to determine how alcohol affects the insulin secretion and viability of human adult pancreatic islets in vitro during a seven-day incubation. Methods. Human pancreatic tissue was digested with Collagenase XI, using a non-automated method. Cultures were incubated in Roswell Park Memorial Institute (RPMI) medium containing alcohol (10 μl of alcohol in 100 ml of medium). Insulin stimulation index (SI) and viability of the islets were determined on the first, third, and seventh day of cultivation. Results. Analysis of the viability of the islets showed that there wasn’t significant difference between the control and the test group. In the test group, viability of the cultures declined with the time of incubation. SI of the test group was higher compared to the control group, by 50% and 25% on the first and third day of cultivation, respectively. On the seventh day, insulin secretion was reduced by 25%. The difference was not statistically significant (p > 0.05). In the test group, significant decline in insulin secretion was found on the third and seventh day of incubation (p ≤ 0.05). Conclusion. Alcohol can increase or decrease insulin secretion of islets cultures, which may result in an inadequate response of pancreatic β-cells to blood glucose, leading to insulin resistance, and increased risk of developing type 2 diabetes. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 41002]
... PHB1 has been reported to promote the survival of many noncancerous cell types, including cardiomyocytes [8,[21][22][23][24][25][26]. Overexpression of PHB1 inhibits the mitochondria-mediated apoptosis pathway in H9c2 cells that is induced by hypoxia. ...
... We found here that in cardiomyocytes, FL3 promotes translocation of PHB1 to mitochondria. This observation is in line with other studies showing that translocation of PHB1 from the nucleus to mitochondria is necessary for cytoprotection in ovarian granulosa cells [23,24], pancreatic β-cells [25], and the retinal epithelium [26]. Accumulation of PHB1 in the mitochondrial membrane can stabilize this membrane, blocking the apoptotic machinery. ...
Aims:
The clinical use of doxorubicin for the treatment of cancer is limited by its cardiotoxicity. Flavaglines are natural products that have both potent anticancer and cardioprotective properties. A synthetic analog of flavaglines, FL3, efficiently protects mice from the cardiotoxicity of doxorubicin. The mechanism underlying this cardioprotective effect has yet to be elucidated.
Methods and results:
Here, we show that FL3 binds to the scaffold proteins prohibitins (PHBs) and thus promotes their translocation to mitochondria in the H9c2 cardiomyocytes. FL3 induces heterodimerization of PHB1 with STAT3, thereby ensuring cardioprotection from doxorubicin toxicity. This interaction is associated with phosphorylation of STAT3. A JAK2 inhibitor, WP1066, suppresses both the phosphorylation of STAT3 and the protective effect of FL3 in cardiomyocytes. The involvement of PHBs in the FL3-mediated cardioprotection was confirmed by means of small interfering RNAs (siRNAs) targeting PHB1 and PHB2. The siRNA knockdown of PHBs inhibits both phosphorylation of STAT3 and the cardioprotective effect of FL3.
Conclusion:
Activation of mitochondrial STAT3/PHB1 complex by PHB ligands may be a new strategy against doxorubicin-induced cardiotoxicity and possibly other cardiac problems.
... Transgenic mice overexpressing PHB in IECs are protected from experimental colitis and exhibit less oxidative stress in the colon [20,22]. This is in agreement with emerging data that suggest a role of PHB in protection against oxidative stress-induced injury in multiple cells types [23][24][25][26][27]. Gene silencing of PHB in cultured IECs induces mitochondrial membrane depolarization and cellular stress pathways including intracellular ROS generation, autophagy, and apoptosis [28]. ...
... Transgenic mice overexpressing PHB in IECs are protected from experimental colitis and exhibit less oxidative stress in the colon [20,22]. This is in agreement with emerging data that suggest a role of PHB in protection against oxidative stress-induced injury in multiple cells types [23][24][25][26][27]. ...
Mitochondrial dysfunction in intestinal epithelial cells (IEC) is thought to precede the onset of inflammatory bowel diseases (IBD). Expression of Prohibitin 1 (PHB), a mitochondrial protein required for optimal electron transport chain (ETC) activity, is decreased in mucosal biopsies during active and inactive IBD. In addition to its activities as a transcription factor, Signal Transducer and Activator of Transcription 3 (Stat3) resides in the mitochondria of cells where phosphorylation at S727 is required for optimal ETC activity and protects against stress-induced mitochondrial dysfunction. Here, we show that PHB overexpression protects against mitochondrial stress and apoptosis of cultured IECs induced by TNFα, which is a pro-inflammatory cytokine involved in IBD pathogenesis. Expression of pS727-Stat3 dominant negative eliminates protection by PHB against TNFα-induced mitochondrial stress and apoptosis. PHB interacts with pS727-Stat3 in the mitochondria of cultured IECs and in colonic epithelium from wild-type mice. Our data suggest a protective role of PHB that is dependent on pS727-Stat3 to prevent mitochondrial dysfunction in IECs. Reduced levels of PHB during IBD may be an underlying factor promoting mitochondrial dysfunction of the intestinal epithelium.
... Mitochondrial dysfunction has a significant role in the development and complications of alcoholic cardiomyopathy (Kim et al., 2001;Vendemiale et al., 2001;Cahill et al., 2005;Hajnoczky et al., 2005;Piano et al., 2007). Chronic alcohol exposure accelerates mitochondrial dysfunction and apoptosis across different organs including the heart, liver and pancreas (Vendemiale et al., 2001;Cahill et al., 2002;Hajnoczky et al., 2005;Lee et al. 2010). Most studies have independently examined the impact of alcohol use on hepatic mitochondria or examined degradation of cardiac function in response to chronic ethanol exposure (Vendemiale et al., 2001;Cahill et al., 2002;Hajnoczky et al., 2005;Lee et al. 2010). ...
... Chronic alcohol exposure accelerates mitochondrial dysfunction and apoptosis across different organs including the heart, liver and pancreas (Vendemiale et al., 2001;Cahill et al., 2002;Hajnoczky et al., 2005;Lee et al. 2010). Most studies have independently examined the impact of alcohol use on hepatic mitochondria or examined degradation of cardiac function in response to chronic ethanol exposure (Vendemiale et al., 2001;Cahill et al., 2002;Hajnoczky et al., 2005;Lee et al. 2010). Far fewer studies have examined alcohol's impact on the myocardial mitochondria. ...
Aims:
Alcoholic cardiomyopathy (ACM) presents as decreased myocardial contractility, arrhythmias and secondary non-ischemic dilated cardiomyopathy leading to heart failure. Mitochondrial dysfunction is known to have a significant role in the development and complications of ACM. This study investigated if chronic ethanol feeding promoted myocardial mitochondrial topoisomerase dysfunction as one underlying cause of mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in ACM.
Methods:
The impact of chronic ethanol exposure on the myocardial mitochondria was examined in both neonatal cardiomyocytes using 50 mM ethanol for 6 days and in rats assigned to control or ethanol feeding groups for 4 months.
Results:
Chronic ethanol feeding led to significant (P < 0.05) decreases in M-mode Fractional Shortening, ejection fraction, and the cardiac output index as well as increases in Tau. Ethanol feeding promoted mitochondrial dysfunction as evidenced by significantly decreased left ventricle cytochrome oxidase activity and decreases in mitochondrial protein content. Both in rats and in cultured cardiomyocytes, chronic ethanol presentation significantly increased mtDNA damage. Using isolated myocardial mitochondria, both mitochondrial topoisomerase-dependent DNA cleavage and DNA relaxation were significantly altered by ethanol feeding.
Conclusion:
Chronic ethanol feeding compromised cardiovascular and mitochondrial function as a result of a decline in mtDNA integrity that was in part the consequence of mitochondrial topoisomerase dysfunction. Understanding the regulation of the mitochondrial topoisomerases is critical for protection of mtDNA, not only for the management of alcoholic cardiomyopathy, but also for the many other clinical treatments that targets the topoisomerases in the alcoholic patient.
... Coronary endothelial cells from diabetic mice are characterized by fragmented mitochondria with a downregulation of OPA1 and an upregulation of DRP1 [141]. In addition, Prohibitin (Phb) has been shown to have a protective role in β cells [142]. Interestingly, phb-genetic ablation results in aberrant mitochondrial cristae structure and an increased apoptosis, dependent on increased proteolytic processing of OPA1 [15]. ...
... T cells are activated at the so-called " immunological synapse " between a T cell and an antigen-presenting cell (APC) [146]. Mitochondria usually Alteration in OPA1 processing in β-cells in the pancreas and coronary endothelial cells from diabetic animals [141, 142]. fragment and relocalize at the immunological synapse in close proximity to the plasma membrane to buffer Ca 2+ entrance and to avoid calcium-dependent T-cell inactivation [147]. ...
Mitochondria are key organelles in the cell, hosting essential functions, from biosynthetic and metabolic pathways, to oxidative phosphorylation and ATP production, from calcium buffering to red-ox homeostasis and apoptotic signalling pathways. Mitochondria are also dynamic organelles, continuously fusing and dividing, and their localization, size and trafficking are finely regulated. Moreover, in recent decades, alterations in mitochondrial function and dynamics have been implicated in an increasing number of diseases. In this review, we focus on the relationship clarified hitherto between mitochondrial dynamics and cancer, neurodegenerative and neuroinflammatory diseases.
... PHB is expressed in pancreatic β-cells, and PHB deficiency can significantly reduce glucose-stimulated insulin secretion (GSIS) [67,68]. Supale et al. [16] discovered that a specific deficiency of PHB in β-cells induced βcell loss, impaired insulin secretion, and the failure of glucose homeostasis, and ultimately caused severe diabetes. ...
Prohibitin-2 (PHB2) is a conserved protein in mitochondria that regulates various biological processes, including cell cycle, proliferation, apoptosis, transcription, signal transduction, and mitochondrial ridge morphogenesis. Recently, there has been growing interest in the biological function of PHB2. This article primarily discusses the recent advances in the role of PHB2 in diseases.
... In the present study, loss of architecture of pancreatic acini with widening of spaces was found in group III histopathology. Similar findings were found by Lee et al. [19], who linked acinar damage to ethanolinduced oxidative stress, which causes free radical production leading to peroxidation of the cell membrane's lipid bilayer, and finally membrane disintegration. Dilated interlobular ducts, pyknotic nuclei, and extensive deposition of collagen fibers surrounding pancreatic acini, ducts, and blood vessels were also seen in group III in the current work. ...
... KDEL receptors are also known to interact with prohibitin-1, a multi-functional protein with multiple cellular locales (i.e., nucleus and mitochondria) that is expressed in pancreatic β-cells and may be involved in diabetes [79,80]. KDEL receptors form complexes with prohibitin-1 and prohibitin-1 assists in the retention of KDEL receptors in the cis-Golgi to allow for Src family kinase activation [81]. ...
The KDEL receptor retrieval pathway is essential for maintaining resident proteins in the endoplasmic reticulum (ER) lumen. ER resident proteins serve a variety of functions, including protein folding and maturation. Perturbations to the lumenal ER microenvironment, such as calcium depletion, can cause protein misfolding and activation of the unfolded protein response (UPR). Additionally, ER resident proteins are secreted from the cell by overwhelming the KDEL receptor retrieval pathway. Recent data show that KDEL receptors are also activated during the UPR through the IRE1/XBP1 signaling pathway as an adaptive response to cellular stress set forth to reduce the loss of ER resident proteins. This review will discuss the emerging connection between UPR activation and KDEL receptors as it pertains to ER proteostasis and disease states.
... 1 PHBs proteins are ubiquitous proteins and are associated with a variety of biological processes including cell cycle, apoptosis, and respiration. [2][3][4][5] PHBs have been identified from eukaryotes, fungi, plants, and animals. 6,7 In humans, the PHB proteins act as transcriptional regulators interacting with PSF3, retinoblastoma proteins (Rb), and E2F. ...
The prohibitins (PHB) are SPFH domain-containing proteins found in the prokaryotes to eukaryotes. The plant PHBs are associated with a wide range of biological processes, including senescence, development, and responses to biotic and abiotic stresses. The PHB proteins are identified and characterized in the number of plant species, such as Arabidopsis, rice, maize, and soybean. However, no systematic identification of PHB proteins was performed in Solanum lycopersicum. In this study, we identified 16 PHB proteins in the tomato genome. The analysis of conserved motifs and gene structure validated the phylogenetic classification of tomato PHB proteins. It was observed that various members of tomato PHB proteins undergo purifying selection based on the Ka/Ks ratio and are targeted by four families of miRNAs. Moreover, SlPHB proteins displayed a very unique expression pattern in different plant parts including fruits at various development stages. It was found that SlPHBs processed various development-related and phytohormone responsive cis-regulatory elements in their promoter regions. Furthermore, the exogenous phyto-hormones treatments (Abscisic acid, indole-3-acetic acid, gibberellic acid, methyl jasmonate) salt and drought stresses induce the expression of SlPHB. Moreover, the subcellular localization assay revealed that SlPHB5 and SlPHB10 were located in the mitochondria. This study systematically summarized the general characterization of SlPHBs in the tomato genome and provides a foundation for the functional characterization of PHB genes in tomato and other plant species. ARTICLE HISTORY
... 1 PHBs proteins are ubiquitous proteins and are associated with a variety of biological processes including cell cycle, apoptosis, and respiration. [2][3][4][5] PHBs have been identified from eukaryotes, fungi, plants, and animals. 6,7 In humans, the PHB proteins act as transcriptional regulators interacting with PSF3, retinoblastoma proteins (Rb), and E2F. ...
The prohibitins (PHB) are SPFH domain containing proteins found in the prokaryotes to eukaryotes. The plant PHBs associated with a wide range of biological processes, including senescence, development, and responses to biotic and abiotic stresses. The PHB proteins identified and characterized in number of plant species, such as Arabidopsis, rice, maize, and soybean. However, no systematic identification of PHB proteins was performed in Solanum lycopersicum genome. In this study, we identified 16 PHB proteins in tomato. The analysis of conserved motifs and gene structure validated the phylogenetic classification of tomato PHB proteins. It was observed that various members of tomato PHB proteins undergoes purifying selection based on Ka/Ks ratio and are targeted by four families of miRNAs. Moreover, SlPHB proteins displayed very unique expression pattern in different plant parts including fruits tissues at various development stages. It was found that SlPHBs processed various development-related and phytohormone responsive cis-regulatory elements in their promoter regions. Furthermore, the exogenous phytohormones treatments (ABA, IAA, GA, MeJA) salt, and drought stresses induced the expression of SlPHB. This study systematically summarized the general characterization of SlPHBs in tomato genome and provides a foundation for functional characterization of PHB genes in tomato and other plant species.
... 41 Other exclusively identified protein on the Ti-SLA surfaces was prohibitin, a protein that has roles in fundamental cellular processes, such as cell proliferation, 42 cell cycle control, 43 differentiation, 44 and immune cell functions like; protection from oxidative stress. 45 On the other hand, similitudes were also found on the protein ...
This study reports the differences in the protein composition of salivary pellicles formed under in situ conditions on two Titanium (Ti) surfaces, with different roughness and wettability. Smooth pretreatment Ti surfaces (Ti‐PT) with an average roughness (Ra) of 0.45 μm and a water contact angle (WCA) of 92.4°, as well as a more rough sandblasted, large grit, acid‐etched treatment Ti surfaces (Ti‐SLA) with a Ra of 3.3 μm and WCA of 131.8°, were tested. The salivary pellicles were quantitatively analyzed by bicinchoninic acid assays, and the protein identification was performed by Nano‐LC–MS/MS (nano mass spectrometry). Protein levels of 2.5, and 9.1 μg/ml were quantified from the detached salivary pellicle formed on the Ti‐PT and Ti‐SLA surfaces, respectively. Using Nano‐LC–MS/MS, a total of 597 proteins were identified on all the substrates tested; 43 proteins were identified only on the Ti‐PT, and 226 proteins were adsorbed solely on the Ti‐SLA substrates. The physicochemical characteristics of the Ti implant surfaces modified the amount and the identity of the salivary proteome of the pellicles formed, confirming the high selectivity of the protein pellicle formed on a surface once is exposed in the oral cavity.
... It is reported that oxidative stress induces deleterious effects in pancreatic β-cells [81,82]. However, PHB expression in pancreatic β-cells is protective against oxidative stress and apoptosis [83]. Furthermore, we previously reported, IL-1β, a pro-inflammatory cytokine induces ER stress and mitochondrial-mediated apoptosis in pancreatic β-cells. ...
The role of mitochondria in apoptosis is well known; however, the mechanisms linking mitochondria to the proapoptotic effects of proinflammatory cytokines, hyperglycemia, and glucolipotoxicity are not completely understood. Complex Ca2+ signaling has emerged as a critical contributor to these proapoptotic effects and has gained significant attention in regulating the signaling processes of mitochondria. In pancreatic β-cells, Ca2+ plays an active role in β-cell function and survival. Prohibitin (PHB), a mitochondrial chaperone, is actively involved in maintaining the architecture of mitochondria. However, its possible interaction with Ca2+-activated signaling pathways has not been explored. The present review aims to examine potential crosstalk between Ca2+ signaling and PHB function in pancreatic β-cells. Moreover, this review will focus on the effects of cytokines and glucolipotoxicity on Ca2+ signaling and its possible interaction with PHB. Improved understanding of this important mitochondrial protein may aid in the design of more targeted drugs to identify specific pathways involved with stress-induced dysfunction in the β-cell.
... It is reported that oxidative stress induces deleterious effects in pancreatic β-cells [81,82]. However, PHB expression in pancreatic β-cells is protective against oxidative stress and apoptosis [83]. Furthermore, we previously reported, IL-1β, a pro-inflammatory cytokine induces ER stress and mitochondrial-mediated apoptosis in pancreatic β-cells. ...
A putative Prohibitin-Calcium nexus in β-cell mitochondria and Diabetes.
... 12 PHB1 translocates to the plasma membrane after undergoing certain PTMs. 13 Also, different stimuli induce PHB1 nuclear-mitochondrial shuttling, like ethanol treatment in b-cells, 14 estrogen in prostate cancer cells, 15 and camptothecin in breast cancer cells. 12 Nonetheless, the mechanisms regulating PHB1's intracellular trafficking are not fully understood. ...
Prohibitin 1 is an evolutionary conserved and ubiquitously expressed protein that exerts different biological functions depending on its subcellular localization. The role of prohibitin 1 in liver cancer is controversial as it can be pro- or anti-tumorigenic. However, most of the studies to date have described prohibitin 1 primarily as a tumor suppressor in the liver. Its deficiency sensitizes the liver to cholestatic liver injury, non-alcoholic fatty liver disease, inflammatory insults, and cancer. Liver-specific Phb1-knockout mice spontaneously develop hepatocellular carcinoma, Phb1 heterozygotes are more susceptible to develop cholangiocarcinoma, and the majority of human hepatocellular carcinomas and cholangiocarcinomas have reduced prohibitin 1 expression. Consistent with a tumor suppressive role in the liver, prohibitin 1 negatively regulates proliferation in hepatocytes and human hepatocellular carcinoma and cholangiocarcinoma cell lines, and multiple oncogenic signaling pathways are activated when prohibitin 1 is deficient. Although best known as a mitochondrial chaperone, prohibitin 1 can protect the liver by mitochondrial-independent mechanisms. This review summarizes what’s known about prohibitin 1’s role in liver pathology, with the focus on hepatoprotection and carcinogenesis.
Impact statement
This review summarizes the last decades of research on PHB1 in liver pathobiology. PHB1 is a key player for liver health as it is hepatoprotective and tumor suppressive. We highlight the importance of PHB1’s subcellular localization, post-translational modifications, and interacting proteins as major determinants of PHB1 cytoprotective function and anti-tumor activity in the liver.
... Total protein was isolated using mammalian protein extract buffer (28-9712-79, GE Life Sciences, Little Chalfont, UK) containing protease inhibitor cocktail (P8340, Sigma, St. Louis, MO, USA). The cytosol and nuclear fraction were prepared as described previously [55,56]. An equal amount of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred on polyvinylidene fluoride membranes. ...
Dipeptidyl peptidase 4 (DPP4) inactivates incretin hormone glucagon-like peptide-1. DPP4 inhibitors may exert beneficial effects on diabetic nephropathy (DN) independently of glycemic control; however, the mechanisms underlying are not fully understood. Here, we investigated the mechanisms of the beneficial effects of DPP4 inhibition on DN using DPP4-deficient (DPP4-def) rats and rat mesangial cells.
Blood glucose and HbA1c significantly increased by streptozotocin (STZ) and no differences were between WT-STZ and DPP4-def-STZ. The albumin level in urine decreased significantly and the albumin/creatinine ratio decreased slightly in DPP4-def-STZ. The glomerular volume in DPP4-def-STZ significantly decreased compared with that of WT-STZ. Advanced glycation end products formation, receptor for AGE (RAGE) protein expression, and its downstream inflammatory cytokines and fibrotic factors in kidney tissue, were significantly suppressed in the DPP4-def-STZ compared to the WT-STZ with increasing glyoxalase-1 (GLO-1) expression responsible for the detoxification of methylglyoxal (MGO). In vitro, exendin-4 suppressed MGO-induced AGEs production by enhancing the expression of GLO-1 and nuclear factor-erythroid 2 p45 subunit-related factor 2, resulting in decreasing pro-inflammatory cytokine levels. This effect was abolished by GLO-1 siRNA.
Our data suggest that endogenously increased GLP-1 in DPP4-deficient rats contributes to the attenuation of DN partially by regulating AGEs formation via upregulation of GLO-1 expression.
... Some of the pleiotropic attributes of PHB is also shared by its homologous protein PHB2 [12,18], including a number of phosphorylation sites that have been implicated in their cell signaling functions and intracellular trafficking [100,101]. For examples, PHBs have been shown to play a crucial role in pancreatic beta cell function [48,102] and in the maturation of T cells [18]. Despite similarities between PHB and PHB2, their functions do not appear to be redundant or compensated by each other. ...
Adipocytes and macrophages, the two major constituents of adipose tissue, exhibit sex differences and work in synergy in adipose tissue physiology and pathophysiology, including obesity-linked insulin resistance and metabolic dysregulation. Sex steroid hormones play a major role in sex differences in adipose tissue biology. However, our knowledge of the molecules that mediate these effects in adipose tissue remains limited. Consequently, it remains unclear whether these effector molecules in different adipose and immune cell types are distinct or if there are also pleiotropic effectors. Recently, a protein named prohibitin (PHB) with cell compartment-and tissue-specific functions has been found to play a role in sex differences in adipose and immune functions. Transgenic (Tg) mouse models overexpressing PHB (PHB-Tg) and a phospho-mutant PHB (mPHB-Tg) from the fatty acid binding protein-4 (Fabp-4) gene promoter display sex-neutral obesity; however, obesity-related insulin resistance and metabolic dysregulation are male-specific. Intriguingly, with aging, the male PHB-Tg mice developed hepatic steatosis and subsequently liver tumors whereas the male mPHB-Tg mice developed lymph node tumors and splenomegaly. Unlike the male transgenic mice, the female PHB-Tg and mPHB-Tg mice remain protected from obesity-related metabolic dysregulation and tumor development. In conclusion, the sex-dimorphic metabolic and immune phenotypes of PHB-Tg and mPHB-Tg mice have revealed PHB as a pleiotropic effector of sex differences in adipose and immune functions. In this mini-review, we will discuss the pleiotropic attributes of PHB and potential mechanisms that may have contributed to the sex-dimorphic metabolic phenotypes in PHB-Tg and mPHB-Tg mice, which warrant future research. We propose that PHB is a prime candidate for a pleiotropic mediator of sex differences in adipose and immune functions in both physiology and pathophysiology, including obesity, insulin resistance, and metabolic dysregulation.
... It is likely that targeting PHB provides a survival advantage for pathogens, which may include a metabolic benefit or survival benefit to escape from the host immune defense, or a combination of both [13,19]. In addition, the impairment of PHB function may inflict oxidative damage, which may be involved in the disease process that has been reported in viral hepatitis [53] and in inflammatory bowel disease [43], as well as in impaired insulin secretion from pancreatic b-cells [54]. More recently, Tortelli et al. [55] have reported that accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death. ...
Immunometabolism, which has important implications in cancer biology, has emerged as a major regulator of different immune cell types. Various factors that integrate metabolic switches within immune cells with signal directed program that promote or inhibit their functions remain largely unidentified. Furthermore, sex differences are known to exist in immune functions and cancer incidences in the body and sex steroid hormones are integral component of these differences. However, factors that mediate such differences, and the potential link between the two fundamental aspects of immune cell biology that contributes to sex differences in health and disease remain unexplored. New evidence derived from novel tissue-specific transgenic mouse models of prohibitin (PHB) has revealed its crucial role in sex differences in adipocyte and macrophage functions and a potential role in endocrine-immune crosstalk. This review provides a point of view on the emerging role of PHB in immune functions with special focus on immunometabolism and on the immunomodulatory effects of sex steroids. We propose that PHB plays a crucial role in integrating cell signaling events with metabolic switches, and may serve as a potential target for cancer immunotherapeutic.
... This is because PHB has important role in mitochondrial function, which is crucial for b-cell function, and PHB level has been reported to be upregulated in murine and human pancreatic b-cell lines as a protective response to oxidative stress. 51 A drastic loss of insulin secretion and a rapid onset of type 2 diabetes in b-cell specific Phb2 knockout mice 52 further confirmed a crucial role of PHB2 and potentially PHB1 in pancreatic b-cell function. PHB2 knockdown often results in a parallel down regulation of its heterodimerizing partner PHB1 and vice versa. ...
The promoter of a gene that is selectively expressed in just a few cell types provides unique opportunities to study: (1) the pleiotropic function of a protein in two different cell types including the cell compartment specific function, and (2) the crosstalk between two cell/tissue types at the systemic level. This is not possible with a ubiquitous or a highly specific gene promoter. The adipocyte protein-2 ( aP2) is one such gene. It is primarily expressed in adipocytes, but also selectively in monocytic macrophages and dendritic cells, among various immune cell types. Thus, the adipocyte protein-2 gene promoter provides an opportunity to simultaneously manipulate adipose and immune functions in a transgenic animal. Prohibitin (PHB) is a pleiotropic protein that has roles in both adipocytes and immune cells. Adipocyte specific functions of prohibitin are mediated through its mitochondrial function, whereas its immune functions are mediated in a phosphorylation-dependent manner. We capitalized on this attribute of prohibitin to explore the crosstalk between adipose and immune functions, and to discern mitochondrial and plasma membrane-associated cell signaling functions of prohibitin, by expressing wild type prohibitin (Mito-Ob) and a phospho-mutant form of prohibitin (m-Mito-Ob) from the protein-2 gene promoter, individually. Both transgenic mice develop obesity in a sex-neutral manner, but develop obesity-related metabolic dysregulation in a male sex-specific manner. Subsequently, the male Mito-Ob mice spontaneously developed type 2 diabetes and liver cancer, whereas the male m-Mito-Ob mice developed lymph node tumors or autoimmune diabetes in a context-dependent manner. This review provides a point of view on the role of prohibitin in mediating sex differences in adipose and immune functions at the systemic level. We discuss the unique attributes of prohibitin and provide a new paradigm in adipose-immune crosstalk mediated through a pleiotropic protein.
Impact statement
Prohibitin (PHB) is ubiquitously expressed and plays a role in adipocyte-immune cell cross-talk. Both male and female transgenic mice expressing wild-type PHB in adipose tissue and in macrophages are obese, but only males develop diabetes and liver cancer. When the mice express PHB mutated on tyrosine-114 in adipocytes and macrophages, both males and females are still obese, but none develops liver cancer; instead, males develop lymph node tumors. Adipocyte specific functions of PHB are mediated through its mitochondrial function, whereas its immune functions are mediated in a phosphorylation-dependent manner. Thus, PHB appears to be an important molecule linking obesity, diabetes, and cancer. In addition, this link appears to be affected by sex steroids. Therefore, targeting PHB may lead to a better understanding of the pathogenesis of obesity, diabetes and cancer.
... Chronic alcohol exposure accelerates mitochondrial dysfunction and apoptosis across different organs including the heart, liver and pancreases [19,64,65,71]. Studies reporting degradation of mitochondrial function have shown significant decreases in mitochondrial protein content both across the whole mitochondrial fraction as well as specific proteins critical for mitochondrial function [52,53,72]. In part due to a decline if the production of mitochondrial proteins, but also a function of the degradation of mitochondrial DNA (mtDNA) [70,73,74]. ...
Alcoholism is the third leading cause of preventable death in the United States. Aside from promoting cardiomyopathies, chronic alcohol consumption is associated with an increased risk of dementia, the development of liver or pancreas failure, and cancers of the oral cavity and pharynx. Although a J-shaped curve for all cause mortality has been identified for average alcohol consumption, irregular heavy drinking also carries significantly greater risks for cardiovascular disease. Alcohol induced cardiovascular disease has a complex multigenic etiology. There is significant variation in the initial presentation of alcoholic cardiomyopathy with diastolic dysfunction possibly being the first indication. Ethanol exposure generates toxic metabolites, primarily acetaldehyde and ROS, which activate several cell signaling systems to alter cell function across many levels. Sudden cardiac death is a known occurrence of alcoholism that may be linked to an arrhythmogenic effect of alcohol. Microscopic and molecular examination of diseased hearts has demonstrated abnormal alterations to various cellular components, including the mitochondria and myofibrils. These studies have shown not only the direct impact on myocardial contractility but also disrupted metabolism that determines the long-term survival of the myocardium. Significant variations in the response to chronic alcohol consumption may be related to unique genotypes that modify the metabolic response to ethanol. Future studies to further characterize the role of different genotypes will help indentify those genotypes are more susceptible to chronic alcohol consumption.
... Several studies have shown that ethanol causes insulin resistance in the liver and skeletal muscle by interfering with insulin signaling [20][21][22] . In addition, several experimental studies have reported that chronic ethanol feeding in rodents causes pancreatic β-cell apoptosis 23,24) and decreases β-cell mass 20,25) . One recent study reported that ethanol causes endoplasmic reticulum stress and impairment of insulin secretion in pancreatic β-cells 26) . ...
Associations between alcohol consumption and type 2 diabetes risk are inconsistent in epidemiologic studies. This study investigated the associations of ADH1B and ALDH2 polymorphisms with fasting blood glucose levels, and the impact of the associations of alcohol consumption with fasting blood glucose levels in Japanese individuals. This cross-sectional study included 907 men and 912 women, aged 35–69 years. The subjects were selected from among the Japan Multi-institutional Collaborative Cohort study across six areas of Japan. The ADH1B and ALDH2 polymorphisms were genotyped by Invader Assays. The ALDH2 Glu504Lys genotypes were associated with different levels of fasting blood glucose in men (P = 0.04). Mean fasting glucose level was positively associated with alcohol consumption in men with the ALDH2 504 Lys allele (Ptrend = 0.02), but not in men with the ALDH2 504Glu/Glu genotype (Ptrend = 0.45), resulting in no statistically significant interaction (P = 0.38). Alcohol consumption was associated with elevated fasting blood glucose levels compared with non-consumers in men (Ptrend = 0.002). The ADH1B Arg48His polymorphism was not associated with FBG levels overall or after stratification for alcohol consumption. These findings suggest that the ALDH2 polymorphism is associated with different levels of fasting blood glucose through alcohol consumption in Japanese men. The interaction of ALDH2 polymorphisms in the association between alcohol consumption and fasting blood glucose warrants further investigation.
... Occurrence of type 2 diabetes leading to myocardial damage is found to be associated with insulin resistance [135], apoptotic death of cardiocyte and interstitial fibrosis, which in turn modulated by mitochondrial dysfunction. Enhanced oxidative stress in pancreas more particularly in pancreatic β-cells due to ethanol results in cellular injury, inhibition of insulin secretion and mitochondrial dysfunction leading to apoptosis [136][137][138]. Overexpression of PHB1 leads to the prevention of pancreatic beta cells undergoing apoptosis induced by ethanol exposure [139]. ...
Background:
Prohibitin (PHB) is overtly conserved evolutionarily and ubiquitously expressed protein with pleiotropic functions in diverse cellular compartments. However, regulation and function of these proteins in different cells, tissues and in various diseases is different as evidenced by expression of these proteins which is found to be reduced in heart diseases, kidney diseases, lung disease, Crohn's disease and ulcerative colitis but this protein is highly expressed in diverse cancers. The mechanism by which this protein acts at the molecular level in different subcellular localizations or in different cells or tissues in different conditions (diseases or normal) has remained poorly understood. There are several studies reported to understand and decipher PHB's role in diseases and/or cancers of ovary, lung, stomach, thyroid, liver, blood, prostrate, gastric, esophagus, glioma, breast, bladder etc. where PHB is shown to act through mechanisms by acting as oncogene, tumor suppressor, antioxidant, antiapoptotic, in angiogenesis, autophagy etc.
Objective:
This review specifically gives attention to the functional role and regulatory mechanism of PHB proteins in cardiovascular health and diseases and its associated implications. Various molecular pathways involved in PHB function and its regulation are analyzed.
Conclusion:
PHB is rapidly emerging as a critical target molecule for cardiovascular signaling. Progress in delineating CVD and mechanisms of PHB in diverse molecular pathways is essential for determining when and how PHB targeted therapy might be feasible. In this regard, new therapies targeting PHB may best be applied in the future together with molecular profiling of CVD for clinical stratification of disease diagnosis and prognosis.
... Ethanol toxicity is well studied in a variety of tissues including liver, brain, kidney, and gastrointestinal tract (Alfonso-Loeches and Guerri 2011; Bujanda 2000; Das Kumar and Vasudevan 2008;Gramenzi et al. 2006). These studies have indicated that ethanol induces apoptosis through oxidative stress, mitochondrial dysfunction, and caspase activation (Chen et al. 2011;Lee et al. 2010;Olney et al. 2002;Ramachandran et al. 2003;Seo et al. 2013). The ethanol can be metabolized through both oxidative and nonoxidative pathways to reduce toxicity. ...
Extracellular adenosine-5′-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y1 receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y1 receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.
... Besides the anti-cancer activities, Roc-A has also been shown to protect primary cells from chemotherapy-induced cell death [17]. Interestingly, Lee et al. reported that PHB is expressed in pancreatic beta-cells and protects against oxidative and proapoptotic effects of ethanol [25]. Luan et al., recently showed that PHB expression levels positively correlate with the maintenance of ERK-driven pancreatic tumorigenesis [26]. ...
Pancreatic cancer ranks No.1 in mortality rate worldwide. This study aims to identify the novel anti-pancreatic cancer drugs. Human pancreatic carcinoma cell lines were purchased from ATCC. CPE-based screening assay was used to examine the cell viability. Patient derived tumor xenografts in SCID mice was established. The Caspase-3 and 7 activities were measured using the Caspase Glo 3/7 Assay kit. Soft agar colony formation assay was used to evaluate the colony formation. Wound healing assay was employed to determine the cell migration. We screened a Chinese herbal product library and found three "hits" that kill cancer cells at nanomolar to micromolar concentrations. One of these compounds, rocaglamide, was found to be potent inhibitors of a wide spectrum of pancreatic cancer cell lines. Furthermore, Rocaglamide reduced the tumor size in a patient-derived pancreatic cancer xenograft mouse model without noticeable toxicity in vivo. Rocaglamide also inhibits pancreatic cancer cell migration and invasion. In conclusion, these data support that Rocaglamide may be a promising anti-pancreatic cancer drug.
... The cells were subsequently incubated for 24 h with ethanol or PBS before inducing differentiation. The appropriate ethanol concentration was selected by generating a dose-response curve of ethanol toxicity using MTT assay (Lee et al. 2010). MTT reduction decreased only by 10% at 80-120 nmol/L ethanol concentrations and by 16% at 140 nmol/L ethanol concentration. ...
Prenatal ethanol exposure causes cellular stress, insulin resistance, and glucose intolerance in adult offspring, with increased gluconeogenesis and reduced muscle glucose transporter-4 (glut4) expression. Impaired insulin activation of Akt and nuclear translocation of histone deacetylases (HDACs) in the liver partly explain increased gluconeogenesis. The mechanism for the reduced glut4 is unknown. Pregnant rats were gavaged with ethanol over the last week of gestation and adult female offspring were studied. Some ethanol exposed offspring was treated with tauroursodeoxycholic acid (TUDCA) for 3 weeks. All these rats underwent intraperitoneal glucose tolerance and insulin tolerance tests. The expression of glut4, HDACs, and markers of endoplasmic reticulum (ER) unfolded protein response (XBP1, CHOP, ATF6) was examined in the gastrocnemius muscle fractions, and in C2C12 muscle cells cultured with ethanol, TUDCA, and HDAC inhibitors. Non-TUDCA-treated rats exposed to prenatal ethanol were insulin resistant and glucose intolerant with reduced muscle glut4 expression, increased ER marker expression, and increased nuclear HDACs, whereas TUDCA-treated rats had normal insulin sensitivity and glucose tolerance with normal glut4 expression, ER marker expression, and HDAC levels. In C2C12 cells, ethanol reduced glut4 expression, but increased ER makers. While TUDCA restored glut4 and ER markers to control levels and HDAC inhibition rescued glut4 expression, HDAC inhibition had no effect on ER markers. The increase in nuclear HDAC levels consequent to prenatal ethanol exposure reduces glut4 expression in adult rat offspring, and this HDAC effect is independent of ER unfolded protein response. HDAC inhibition by TUDCA restores glut4 expression, with improvement in insulin sensitivity and glucose tolerance.
... 4,5 Deletion of prohibitin is lethal, highlighting its essential biological role, and its upregulation is protective for intestinal epithelial cells, cardiomyocytes, and pancreatic-β cells. [6][7][8] Prohibitin is also emerging as a key regulator of neuronal survival. It is upregulated in models of ischemic tolerance and in exercise-induced neuroplasticity, 9,10 suggesting a cytoprotective role in brain. ...
Prohibitin is a multi-functional protein involved in numerous cellular activities. Prohibitin overexpression protects neurons from injury in vitro, but it is unclear whether prohibitin can protect selectively vulnerable hippocampal CA1 neurons in a clinically relevant injury model in vivo and, if so, whether the salvaged neurons remain functional.
A mouse model of transient forebrain ischemia that mimics the brain damage produced by cardiac arrest in humans was used to test whether prohibitin expression protects CA1 neurons from injury. Prohibitin-expressing viral vector was microinjected in mouse hippocampus to upregulate prohibitin.
Prohibitin overexpression protected CA1 neurons from transient forebrain ischemia. The protection was associated with dampened postischemic reactive oxygen species generation, reduced mitochondrial cytochrome c release, and decreased caspase-3 activation. Importantly, the improvement in CA1 neuronal viability translated into an improvement in hippocampal function: prohibitin expression ameliorated the spatial memory deficit induced by ischemia, assessed by the Y-maze test, and restored postischemic synaptic plasticity assessed by long-term potentiation, indicating that the neurons spared form ischemic damage were functionally competent.
These data demonstrate that prohibitin overexpression protects highly vulnerable CA1 neurons from ischemic injury in vivo and suggest that the effect is mediated by reduction of postischemic reactive oxygen species generation and preservation of mitochondrial outer membrane integrity that prevents activation of apoptosis. Measures to enhance prohibitin expression could have translational value in ischemic brain injury and, possibly, other forms of brain injury associated with mitochondrial dysfunction.
... Although rhein shows a similar or even better effect on reducing mouse blood glucose level than rosiglitazone, the underlying mechanism remains unclear. It has been known that mitochondrial fission and fusion modulators, dynaminrelated protein 1 (Drp1) (12), optic atrophy protein 1 (Opa1) (13), prohibitin (14), and mitofusin (15), collectively control the dynamic balance of mitochondria fission and fusion processes and consequent mitochondria functions. Previous studies have demonstrated that Drp1 plays an important role in promoting hyperglycemia-induced apoptosis of b-cells and neurons (12,16,17). ...
Rhein, an anthraquinone compound isolated from rhubarb, has been shown to improve glucose metabolism disorders in diabetic mice. The mechanism underlying the protective effect of Rhein, however, remains unknown. Here, we demonstrate that Rhein can protect the pancreatic β-cells against hyperglycemia-induced cell apoptosis through stabilizing mitochondrial morphology. Oral administration of Rhein for 8 or 16 weeks in db/db mice significantly reduced fasting blood glucose level and improved glucose tolerance. Cell apoptosis assay using both pancreatic sections and cultured pancreatic β-cells indicated that Rhein strongly inhibited β-cell apoptosis. Morphological study showed that Rhein was mainly localized at β-cell mitochondria and Rhein could preserve mitochondrial ultrastructure by abolishing hyperglycemia-induced mitochondrial fission protein dynamin-related protein 1 (Drp1) expression. Western blot and functional analysis confirmed that Rhein protected the pancreatic β-cells against hyperglycemia-induced apoptosis via suppressing mitochondrial Drp1 level. Finally, mechanistic study further suggested that decreased Drp1 level by Rhein might be due to its effect on reducing cellular reactive oxygen species (ROS). Taken together, our study demonstrates for the first time that Rhein can serve as a novel therapeutic agent for hyperglycemia treatment, and Rhein protects pancreatic β-cells from apoptosis by blocking the hyperglycemia-induced Drp1 expression.
... 34 It is well established that heavy ethanol consumption in adults affects adversely pancreatic function and represents a potentially important, modifiable risk factor of type 2 diabetes. [56][57][58] Lee et al. 59 determined that excessive or chronic ethanol consumption leads to oxidative stress and mitochondrial dysfunction in pancreatic b-cells. Furthermore, a study by Nguyen et al. 57 found that ethanol decreases insulin secretion from b-cell lines and isolated murine islets by interfering with muscarinic signaling and PKC activation. ...
Background:
Ethanol consumption during pregnancy can lead to a range of adverse developmental outcomes in children, termed fetal alcohol spectrum disorder (FASD). Central nervous system injury is a debilitating and widely studied manifestation of chronic prenatal ethanol exposure (CPEE). However, CPEE can also cause structural and functional deficits in metabolic pathways in offspring.
Objectives and Methods:
This study tested the hypothesis that CPEE increases whole-body adiposity and disrupts pancreatic structure in guinea pig offspring. Pregnant guinea pigs received ethanol (4 g kg−1 maternal body weight per day) or isocaloric-sucrose/pair-feeding (control) for 5 days per week throughout gestation.
Results:
Male and female CPEE offspring demonstrated growth restriction at birth, followed by a rapid period of catch-up growth before weaning (postnatal day (PD) 1–7). Whole-body magnetic resonance imaging (MRI) in young adult offspring (PD100–140) revealed increased visceral and subcutaneous adiposity produced by CPEE. At the time of killing (PD150–200), CPEE offspring also had increased pancreatic adipocyte area and decreased β-cell insulin-like immunopositive area, suggesting reduced insulin production and/or secretion from pancreatic islets.
Conclusion:
CPEE causes increased adiposity and pancreatic dysmorphology in offspring, which may signify increased risk for the development of metabolic syndrome and type 2 diabetes mellitus.
Aims/Introduction
Mitochondrial damage caused by oxidative stress is a main driver of pancreatic β‐cell dysfunction in the pathogenesis of type 2 diabetes mellitus. Prohibitin2 (PHB2) is a vital inner mitochondrial membrane protein that participates in mitophagy to remove the damaged mitochondria. This study aimed to investigate the role and mechanisms of PHB2‐mediated mitophagy in oxidative stress‐induced pancreatic β‐cell dysfunction.
Materials and Methods
PHB2 and mitophagy‐related protein expression were analyzed by real‐time polymerase chain reaction and western blotting in RINm5F cells treated with H 2 O 2 and islets of diabetic rats. Mitophagy was observed by mitochondrial and lysosome colocalization. RINm5F cells were transfected by phb2 siRNA or overexpression plasmid to explore the role of PHB2 in mitophagy of RINm5F cells. The mechanism of Nrf2 regulating PHB2 was explored by Nrf2 inhibitor and agonist.
Results
The expression of PHB2, mitophagy related protein PINK1, and Parkin were decreased in RINm5F cells incubated with H 2 O 2 and in islets of diabetic rats. Overexpression of PHB2 protected β‐cells from oxidative stress by promoting mitophagy and inhibiting cell apoptosis, whereas transfection with PHB2 siRNA suppressed mitophagy. Furthermore, PHB2‐mediated mitophagy induced by oxidative stress was through the Nrf2/PHB2 pathway in β‐cells. Antioxidant NAC alleviated oxidative stress injury by promoting PHB2‐mediated mitophagy.
Conclusion
Our study suggested that PHB2‐mediated mitophagy can protect β‐cells from apoptosis via the Nrf2/PHB2 pathway under oxidative stress. Antioxidants may protect β‐cell from oxidative stress by prompting PHB2‐mediated mitophagy. PHB2‐mediated mitophagy as a potential mechanism takes part in the oxidative stress induced β‐cell injury.
Although Cd accumulation varies among rice varieties is recognized, the underlying mechanisms are not well clarified. In this study, comparative transcriptome analysis were performed by hydroponic culture system with two rice varieties, Y1540 (high Cd accumulator) and Y15 (low Cd accumulator) under 20 μM Cd stress. Results revealed 17,320 differentially expressed genes (DEGs) in roots of Y15 (7,655 upregulated and 9,665 downregulated) and 17,386 DEGs in roots of Y1540 (8,823 upregulated and 8,563 downregulated) expose to 20 μM Cd stress. Gene ontology (GO) analysis enriched 24 and 26 terms in Y15 and Y1540 respectively, including 23 common terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed 27 and 28 significant pathways in Y15 and Y1540 respectively, with 19 common pathways. Different responses to Cd stress between cultivars were not only reflected in differently enriched GO terms and KEGG pathways but also in different DEGs of 23 common GO terms and significant sequences represented by p-values of 19 common KEGG pathways. Both cultivars resist Cd through common processes with different weights; hence glutathione metabolism, mineral absorption, biosynthesis of secondary metabolites, and degradation of aromatic compounds could be playing a more important role in Y1540, whereas ribosome biogenesis in eukaryotes, mismatch repair, aminoacyl-tRNA biosynthesis, and the cell cycle maybe playing a more important role in Y15. Weighted gene co-expression network analysis (WGCNA) showed that five and three modules were clustered in Y15 and Y1540, respectively, with yellow and brown modules in Y15 and brown modules in Y1540 being significantly related to Cd stress. Further analysis showed that most of hub genes in Y15 were related to signal transduction or transcription factors, while most of hub genes in Y1540 were related to binding, metabolic, and secondary metabolic processes, which demonstrated their different response patterns at transcriptomic level to Cd stress.
SIRT1 has been proposed to enhance insulin secretion in β-cell through repressing the expression of uncoupling protein2 (UCP2), but whether ethanol-induced β-cell dysfunction is mediated by the disrupted SIRT1-UCP2 axis remains unknown. This study was conducted to explore the underlying mechanisms by which ethanol resulted in β-cell dysfunction and the potential protective effects of resveratrol in this process. INS-1 cells (rat pancreatic β-cell line) were cultured with ethanol in the presence or absence of resveratrol (2.5, 12.5 μmol/L). The results showed that ethanol exposure reduced glucose-stimulated insulin secretion, ATP production and SIRT1 expression but increased UCP2 expression, while supplementation with resveratrol restored the function of INS-1 cell by upregulating SIRT1 and inhibiting UCP2. Moreover, the critical role of SIRT1-UCP2 axis was further supported by the results that SIRT1 activator SRT1720 reversed ethanol-induced impairment of glucose-stimulated insulin secretion by decreasing UCP2, while SIRT1 inhibitor Ex527 abolished the beneficial effects of resveratrol. Meanwhile, NAD+ booster nicotinamide mononucleotide also counteracted the deleterious effects of ethanol by increasing SIRT1, suggesting the regulation of SIRT1-UCP2 axis may be associated with cellular NAD+/NADH ratio. In conclusion, our observations imply that ethanol induces impaired insulin secretion from INS-1 cell through disrupting SIRT1-UCP2 axis, while resveratrol may reverse this process by augmenting SIRT1 and inhibiting UCP2.
Influenza virus infection is associated with type 1 diabetes (T1DM), but its pathogenesis remains unclear. Here, our study found that one of the monoclonal antibodies against H1N1 influenza virus hemagglutinin(HA) cross-reacted with human pancreatic tissue and further demonstrated that it binded to rat islet β-cells. We immunoprecipitated islet protein with this cross-reactive antibody and identified the bound antigen as prohibitin by mass spectrometry. We then expressed the prohibitin protein in bacteria and confirmed the antibody binding to prohibitin by Western blot. We also verified the cross-reactivity of the antibody by prohibitin-siRNA transfection in islet beta cells. We conclude that prohibitin is an autoantigen that cross-reacts with influenza virus HA. The correlation between the autoantigen prohibitin and type 1 diabetes remains to be investigated.
Nous avons développé trois accès synthétiques performants à des cyclopentènones fonctionnalisées en exploitant des réactivités inattendues que nous avons découvertes. Nous avons aussi effectué la première synthèse d’isostères des flavaglines substitués par un groupement formylamino ou mésylamino en position 1b et ainsi démontré l’importance de l’hydroxyl en cette position pour la cytotoxicité de ces composés. De plus, nous avons aussi contribué à l’exploration du potentiel thérapeutique des flavaglines et d’un autre ligand des prohibitines, la fluorizoline, dans le traitement des cancers et de l’inflammation chronique des intestins, ainsi que dans la prévention des effets adverses des chimiothérapies au niveau cardiaque.
Prohibitin2 (PHB2), a potential tumor suppressor protein, plays important roles in inhibition of cell cycle progression, transcriptional regulation, apoptosis and the mitochondrial respiratory chain. To explore its potential roles in crustaceans' immune responses we have identified and characterized LvPHB2, a 891 bp gene encoding a 297 amino acids protein in the shrimp Litopenaeus vannamei. Expression analyses showed that LvPHB2 is expressed in all examined tissues, and largely present in cytoplasm, correlating with its known anti-oxidation function in mitochondria. Luciferase reporter assays showed that over-expression of LvPHB2 could activate the p53 pathway, indicating that it might participate in apoptosis regulation. Quantitative real-time PCR revealed that infection with Vibrio alginolyticus induces its up-regulation in hepatopancreas. Moreover, RNAi knock-down of LvPHB2 in vivo raises mortality rates of L. vannamei infected by V. alginolyticus, and affects expression of STAT3, Caspase3 and p53 genes. We found significantly higher reactive oxygen species production, DNA damage and apoptosis rates in LvPHB2-silenced shrimp challenged with V. alginolyticus than in controls injected with a Green Fluorescent Protein-silencing construct. Our results suggest that LvPHB2 plays a vital role in shrimp responses to V. alginolyticus infection through its participation in regulation of oxidants and apoptosis.
Oxidative stress mediated by reactive oxygen species (ROS) contributes to renal tubular atrophy and fibrosis following renal transplantation. Studies have shown that mitochondrial chaperone prohibitin (PHB) has antioxidant effects. Here we used the human renal proximal tubule epithelial cell line, HK-2 cells as an in vitro model to assess the role of PHB in hydrogen peroxide (H2O2)-induced renal tubular oxidative injury. Our results showed that H2O2 treatment inhibited PHB expression in a time-dependent manner in HK-2 cells. PHB overexpression could protect cell from oxidative stress-induced injury by inhibiting H2O2-induced cell apoptosis, intracellular ROS generation and promoting endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, and glutathione. Furthermore, oxidative stress-induced cell injury were suppressed in PHB-overexpressed tubule epithelial cells through mitochondria-mediated pathway, including inhibition of mitochondrial uncoupling protein 2 (UCP2) and Bax expression, mitochondrial ROS production, promotion of ATP production and Bcl-2 expression, inhibition of mitochondrial membrane potential loss and release of cytochrome c from mitochondria to cytoplasma and followed by caspase 3 activation. Meanwhile, inhibition of PHB expression by small interference RNA resulted in less resistance of HK-2 cells to H2O2 toxicity as shown in decreased cell viability, increased apoptosis, ROS production and mitochondrial dysfunction. These data indicated that PHB protected the tubule epithelial cells from oxidative stress-induced damage through the inhibition of oxidative damage, mitochondria dysfunction and ultimately inhibition of cell apoptosis, and that increasing PHB content in mitochondria constituted a new therapeutic target for transient ischemic injury and chronic allograft nephropathy (CAN) following renal transplantation.
Human eukaryotic prohibitin (prohibitin-1 and prohibitin-2) is a membrane protein with different cellular localizations. It is involved in multiple cellular functions, including energy metabolism, proliferation, apoptosis, and senescence. The subcellular localization of prohibitin may determine its functions. Membrane prohibitin regulate the cellular signaling of membrane transport, nuclear prohibitin control transcription activation and the cell cycle, and mitochondrial prohibitin complex stabilize the mitochondrial genome and modulate mitochondrial dynamics, mitochondrial morphology, mitochondrial biogenesis, and the mitochondrial intrinsic apoptotic pathway. Moreover, prohibitin can translocates into the nucleus or the mitochondria under apoptotic signals and the subcellular shuttling of prohibitin is necessary for apoptosis process. Apoptosis is the process of programmed cell death that is important for the maintenance of normal physiological functions. Consequently, any alteration in the content, post-transcriptional modification (i.e. phosphorylation) or the nuclear or mitochondrial translocation of prohibitin may influence cell fate. Understanding the mechanisms of the expression and regulation of prohibitin may be useful for future research. This review provides an overview of the multifaceted and essential roles played by prohibitin in the regulation of cell survival and apoptosis.
The highly conserved protein prohibitin 2 (PHB2) has been implicated as a cell-surface receptor in the regulation of proliferation, apoptosis, transcription, and mitochondrial protein folding. In the present study, we identified a Lampetra morii homologue of PHB2, Lm-PHB2, showing greater than 61.8% sequence identity with its homologues. Phylogenetic analysis indicated that the position of Lm-PHB2 is consistent with lamprey phylogeny. Expression of the Lm-PHB2 protein was nearly equivalent in the heart, liver, kidneys, intestines, and muscles of normal lampreys. However, the Lm-PHB2 protein was down-regulated in the myocardia of lampreys challenged for 5 days with adriamycin (Adr), followed by a significant up-regulation 10 days after treatment. In vitro, recombinant Lm-PHB2 (rLm-PHB2) protein could significantly enhance the H2O2-induced oxidative stress tolerance in Chang liver (CHL) cells. Further mechanism studies indicated that the nucleus-to-mitochondria translocation of Lm-PHB2 was closely involved in the oxidative stress protection. Our results suggests that the strategies to modulate Lm-PHB2 levels may constitute a novel therapeutic approach for myocardial injury and liver inflammatory diseases, conditions in which oxidative stress plays a critical role in tissue injury and inflammation.
Copyright © 2014. Published by Elsevier Ltd.
Alcohol overindulgence is a risk factor of type 2 diabetes mellitus. However, the mechanisms by which alcohol overindulgence damages glucose metabolism remain unclear. Pancreatic islet β-cells are endowed with type-A γ-aminobutyric acid receptor (GABAAR) mediated autocrine signaling mechanism, which regulates insulin secretion and fine-tunes glucose metabolism. In neurons GABAAR is one of the major targets for alcohol. This study investigated whether ethanol alters glucose metabolism by affecting GABAAR signaling in pancreatic β-cells. Blood glucose level of test mice was measured using a blood glucose meter. Insulin secretion by the pancreatic β-cell line INS-1 cells was examined using a specific insulin ELISA kit. Whole-cell patch-clamp recording was used to evaluate GABA-elicited current in INS-1 cells. Western blot and immunostaining were used to measure the expression of GABAAR subunits in mouse pancreatic tissues or in INS-1 cells. Intraperitoneal (i.p.) administration of ethanol (3.0 g/kg body weight) to mice altered glucose metabolism, which was associated with decreased expression of GABAAR α1- and δ- subunits on the surface of pancreatic β-cells. Acute treatment of cultured INS-1cells with ethanol (60 mM) decreased the GABA-induced current and reduced insulin secretion. In contrast, treating INS-1 cells with GABA (100 μM) largely prevented the ethanol-induced reduction of insulin release. Importantly, pre-treating mice with GABA (i.p., 1.5 mg/kg body weight) partially reversed ethanol-induced impairment of glucose homeostasis in mice. Our data suggest a novel role of pancreatic GABA signaling in protecting pancreatic islet β-cells from ethanol-induced dysfunction.
Prohibitins (PHBs) are highly conserved proteins in species ranging from prokaryotes to eukaryotes. Plant PHBs have been implicated in various cellular processes including development, senescence and stress responses. Although PHBs have been investigated in several plant species including Arabidopsis and tobacco, no systematic gene family analysis has been carried in maize. In the present study, 16 putative PHB genes have been identified. Analysis of the conserved protein motifs and gene structures has revealed high levels of conservation within the phylogenetic subgroups. Published microarray database showed that most maize PHB genes exhibited different expression levels in different tissues and developmental stages. Cis-elements analysis showed that ZmPHB2 and ZmPHB12 may play important roles in plant development. Taken together, we provide a comprehensive bioinformatics analysis of the PHB gene family in maize genome and our data provide an important foundation for further functional study of this gene family in maize.
(+)-Usnic acid (UA) has been known to be a strong uncoupler, and mitochondrial and endoplasmic reticulum (ER)-related stresses are suggested to be involved in the mechanism of hepatotoxicity. However, it has not been clarified whether UA causes toxicity in other mitochondria-rich organs such as the heart. We elucidated whether UA induces cardiotoxicity and its mechanism. UA was orally administered to rats for 14 days, and laboratory and histopathological examinations were performed in conjunction with toxicogenomic analysis. As a result, there was no alteration in blood chemistry, whereas cytoplasmic rarefaction of myocardium was observed microscopically. This finding corresponded to the swollen mitochondria observed ultrastructurally. Immunohistochemically, expression of prohibitin, indicating mitochondrial imbalance, increased in the sarcoplasmic area. Toxicogenomic analysis highlighted the upregulation of gene groups consisting of oxidative stress, ER stress, and amino acid limitation. Interestingly, the number of upregulated genes was larger in the amino acid limitation-related gene group than that in other groups, implying that amino acid limitation might be one of the sources of oxidative stress, not only mitochondria and ER-originated stresses. In conclusion, the heart was manifested to be one of the target organs of UA. Mitochondrial imbalance with complex stresses may be involved in the toxic mechanism.
Abstract Prohibitin (PHB), appearing to be a negative regulator of cell proliferation and to be a tumor suppressor, has been connected to diverse cellular functions including cell cycle control, senescence, apoptosis and the regulation of mitochondrial activities. It is a growth regulatory gene that has pleiotropic functions in the nucleus, mitochondria and cytoplasmic compartments. However, in different tissues/cells, the expression of PHB was different, such as that it was increased in most of the cancers, but its expression was reduced in kidney diseases. Signaling pathways might be very important in the pathogenesis of diseases. This review was performed to provide a relatively complete signaling pathways flowchart for PHB to the investigators who were interested in the roles of PHB in the pathogenesis of diseases. Here, we review the signal transduction pathways of PHB and its role in the pathogenesis of diseases.
Although originally identified as putative negative regulators of the cell cycle, recent studies have demonstrated that the PHB proteins act as a chaperone in the assembly of subunits of mitochondrial respiratory chain complexes. The two PHB proteins, Phblp and Phb2p, are located in the mitochondrial inner membrane where they form a large complex that represents a novel type of membrane-bound chaperone. On the basis of its native molecular weight, the PHB-complex should contain 12-14 copies of both Phblp and Phb2p. The PHB complex binds directly to newly synthesised mitochondrial translation products and stabilises them against degradation by membrane-bound metalloproteases belonging to the family of mitochondrial triple-A proteins. Sequence homology assigns Phb1p and Phb2p to a family of proteins which also contains stomatins, HflKC, flotillins and plant defence proteins. However, to date only the bacterial HflKC proteins have been shown to possess a direct functional homology with the PHB complex. Previously assigned actions of the PHB proteins, including roles in tumour suppression, cell cycle regulation, immunoglobulin M receptor binding and apoptosis seem unlikely in view of any hard evidence in their support. Nevertheless, because the proteins are probably indirectly involved in ageing and cancer, we assess their possible role in these processes. Finally, we suggest that the original name for these proteins, the prohibitins, should be amended to reflect their roles as proteins that hold badly formed subunits, thereby keeping the nomenclature already in use but altering its meaning to reflect their true function more accurately.
Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H(2)O(2)) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury.
The two major classes of antigen receptors on murine B lymphocytes, mIgM and mIgD, are both contained in a complex with two additional molecules, Ig-alpha and Ig-beta, which permit signal transduction. Accordingly, early biochemical events after antigen binding to either receptor are similar; biological effects, however, are different. Here, we describe three newly discovered intracellular proteins of 32, 37 and 41 kDa molecular mass, that are non-covalently associated with mIgM, but not with mIgD. These proteins coprecipitate with mIgM in Triton X-100 and Nonidet P-40, but not in digitonin lysates. In addition, mIgM is to some extent associated with 29 and 31 kDa proteins that are predominantly associated with mIgD (see accompanying paper). Amino acid sequencing of p32 and p37 identified p32 as mouse prohibitin; this was corroborated by Western blot analysis with antibodies specific for rat prohibitin. p37 is a newly discovered protein. cDNA clones for both proteins were isolated and sequenced. The deduced amino acid sequence of p32 is identical to that of rat prohibitin. p37 is highly homologous to p32. Since prohibitin was identified as an inhibitor of cell proliferation, its association with mIgM, but not mIgD, could explain the different biological events elicited after engagement of each receptor.
This study demonstrates that rat islet beta cells constitutively express an apoptotic program which is activated when mRNA or protein synthesis is blocked. Apoptotic beta cells were detectable by electron microscopy after treatment with actinomycin D or cycloheximide. With a fluorescence microscopic assay both agents were found to increase the number of apoptotic beta cells dose- and time-dependently, up to 70% after 1 wk of culture; virtually no apoptotic beta cells occurred in control preparations or in conditions leading to primary necrosis. Thus, survival of beta cells seems dependent on synthesis of proteins which suppress an endogenous suicide program. This mechanism explains earlier observed effects of glucose on survival of cultured beta cells. Glucose is known to dose-dependently increase the percentage of beta cells in active biosynthesis and the percentage that survives during culture. It is now demonstrated that the glucose-induced survival of beta cells cultured for 1 wk results from a dose-dependent reduction in the percentage of beta cells dying in apoptosis (49% at 3 mM glucose, 40% at 6 mM, 9% at 10 mM). Thus, intercellular differences in glucose sensitivity appear responsible for the heterogeneity in beta cell sensitivity to apoptotic conditions. These data indicate that glucose promotes survival of beta cells by activating synthesis of proteins which suppress apoptosis. The present model allows for further investigation of the regulation of apoptosis in beta cells and the identification of agents which induce or prevent beta cell death.
Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity.
Reactive oxygen species are involved in a diversity of biological phenomena such as inflammation, carcinogenesis, aging, and atherosclerosis. We and other investigators have shown that the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker for oxidative stress, is increased in either the urine or the mononuclear cells of the blood of type 2 diabetic patients. However, the association between type 2 diabetes and oxidative stress in the pancreatic beta-cells has not been previously described. We measured the levels of 8-OHdG and 4-hydroxy-2-nonenal (HNE)-modified proteins in the pancreatic beta-cells of GK rats, a model of nonobese type 2 diabetes. Quantitative immunohistochemical analyses with specific antibodies revealed higher levels of 8-OHdG and HNE-modified proteins in the pancreatic beta-cells of GK rats than in the control Wistar rats, with the levels increasing proportionally with age and fibrosis of the pancreatic islets. We further investigated whether the levels of 8-OHdG and HNE-modified proteins would be modified in the pancreatic beta-cells of GK rats fed with 30% sucrose solution or 50 ppm of voglibose (alpha-glucosidase inhibitor). In the GK rats, the levels of 8-OHdG and HNE-modified proteins, as well as islet fibrosis, were increased by sucrose treatment but reduced by voglibose treatment. These results indicate that the pancreatic beta-cells of GK rats are oxidatively stressed, and that chronic hyperglycemia might be responsible for the oxidative stress observed in the pancreatic beta-cells.
The retinoblastoma tumor suppressor protein and its family members, p107 and p130, are major regulators of the mammalian cell cycle. They exert their growth suppressive effects at least in part by binding the E2F family of transcription factors and inhibiting their transcriptional activity. Agents that disrupt the interaction between Rb family proteins and E2F promote cell proliferation. Here we describe the characterization of a novel interaction between Rb family proteins and a potential tumor suppressor protein, prohibitin. Prohibitin physically interacts with all three Rb family proteins in vitro and in vivo, and was very effective in repressing E2F-mediated transcription. Prohibitin could inhibit the activity of E2Fs 1, 2, 3, 4 and 5, but could not affect the activity of promoters lacking an E2F site. Surprisingly, prohibitin-mediated repression of E2F could not be reversed by adenovirus E1A protein. A prohibitin mutant that could not bind to Rb was impaired in its ability to repress E2F activity and inhibit cell proliferation. We believe that prohibitin is a novel regulator of E2F activity that responds to specific signaling cascades.
Prolonged poor glycaemic control in patients with Type II (non-insulin-dependent) diabetes mellitus often causes pancreatic beta-cell dysfunction accompanied by decreases in insulin biosynthesis and beta-cell proliferation. This is well known as a clinical concept called glucose toxicity. Whereas oxidative stress is provoked under diabetic conditions, we examined the possible implication of cyclin-dependent kinase (Cdk) inhibitor p21 (WAF1/CIP1/Sdi1) in beta-cell dysfunction mediated by oxidative stress.
Oxidative stress was induced in isolated rat pancreatic islet cells by treatment with H(2)O(2) and mRNA expression of p21 and insulin was examined by northern blot analyses. Also, the expression of p21 and insulin mRNA was examined in Zucker diabetic fatty rat. In islet cells p21 was overexpressed using adenovirus and its effect on insulin gene transcription was examined.
When oxidative stress was charged on isolated rat pancreatic islet cells, p21 mRNA expression was induced whereas insulin mRNA was decreased. Also, when diabetes developed in Zucker diabetic fatty rats, p21 expression was induced and the insulin mRNA expression was reduced. As support for the implication of p21 in impairment of beta-cell function, the p21 overexpression in the islet cells suppressed the insulin gene transcription.
The expression of cyclin-dependent kinase inhibitor p21, which can be induced by oxidative stress, increases in pancreatic islet cells upon development of diabetes. By suppressing cell proliferation and insulin biosynthesis, the p21 induction is likely to be implicated in the beta-cell glucose toxicity.
Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes adverse alterations in beta cell function, a phenomenon termed glucose toxicity and one that may play a secondary pathogenic role in type 2 diabetes. However, no mechanism of action has been definitively identified for glucose toxicity in beta cells. To ascertain whether chronic oxidative stress might play a role, we chronically cultured the beta cell line, HIT-T15, in medium containing 11.1 mM glucose with and without the antioxidants, N-acetyl-L-cysteine (NAC) or aminoguanidine (AG). Addition of NAC or AG to the culture medium at least partially prevented decreases in insulin mRNA, insulin gene promoter activity, DNA binding of two important insulin promoter transcription factors (PDX-1/STF-1 and RIPE-3b1 activator), insulin content, and glucose-induced insulin secretion. These findings suggested that one mechanism of glucose toxicity in the beta cell may be chronic exposure to reactive oxygen species, i.e., chronic oxidative stress. To ascertain the effects of these drugs on diabetes, NAC or AG was given to Zucker diabetic fatty rats, a laboratory model of type 2 diabetes, from 6 through 12 weeks of age. Both drugs prevented a rise in blood oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde + 4-hydroxy-2-nonenal), and partially prevented hyperglycemia, glucose intolerance, defective insulin secretion as well as decrements in beta cell insulin content, insulin gene expression, and PDX-1 (STF-1) binding to the insulin gene promoter. We conclude that chronic oxidative stress may play a role in glucose toxicity, which in turn may worsen the severity of type 2 diabetes.
Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse-chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.
Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated beta-cell destruction. In insulin-producing RINm5F cells, overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined whether overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Zn superoxide dismutase (SOD) can provide protection for bioengineered RINm5F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F control cells to interleukin-1beta (IL-1beta) alone or a combination of IL-1beta, tumor necrosis factor-alpha, and gamma-interferon resulted in a time- and concentration-dependent decrease of cell viability in the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1beta alone caused only a moderate reduction of viability in the range of 25%, the cytokine mixture induced a significant loss of viability of >75%. This increased toxicity of the cytokine mixture compared with that of IL-1beta alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic synergism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn SOD protected against toxicity of the cytokine mixture but not against that of IL-1beta alone. The reduction of cytokine-mediated toxicity was evident also because of an increased proliferation rate and a drastic decrease in the cell death rate. The improved antioxidant defense status did not prevent the activation of iNOS after cytokine exposure. However, RINm5F cells overexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn SOD overexpression was apparently because of an inactivation of ROS generated in the signal cascades of the cytokines. Overexpression of cytoprotective enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.
Vitamin D3 inhibits cell growth and induces apoptosis in several human cancer lines in vitro and in vivo. However, little is known about the molecular events involved in vitamin D3-induced apoptosis. Here, we demonstrate that the growth-promoting/pro-survival signaling molecule mitogen-activated protein
kinase kinase (MEK) is cleaved in a caspase-dependent manner in murine squamous cell carcinoma (SCC) cells induced to undergo
apoptosis by treatment with vitamin D3. Cleavage resulted in nearly complete loss of full-length MEK and ERK1/2 phosphorylation. ERK1/2 expression was affected
only slightly. The phosphorylation and expression of Akt, a kinase regulating a second cell survival pathway, was also inhibited
after treatment with vitamin D3. However, the pro-apoptotic signaling molecule MEKK-1 was up-regulated in both apoptotic and non-apoptotic cells with greater
induction and partial N-terminal proteolysis of MEKK-1 observed in apoptotic cells. In contrast to vitamin D3, cisplatin and etoposide down-regulated Akt levels only modestly, did not promote significant loss of MEK expression, and
did not up-regulate MEKK-1. We propose that vitamin D3 induces apoptosis in SCC cells by a unique mechanism involving selective caspase-dependent MEK cleavage and up-regulation
of MEKK-1. Additional evidence is provided that vitamin D3-induced apoptosis may be mediated via p38 MAPK.
Evidence regarding the association between alcohol consumption and type 2 diabetes risk remains inconsistent, particularly with regard to male-female differences. The authors conducted a prospective study of type 2 diabetes risk associated with alcohol consumption in a cohort of 12,261 middle-aged participants of the Atherosclerosis Risk in Communities Study (1990-1998), who were followed between 3 and 6 years. Alcohol consumption at baseline was characterized into lifetime abstainers, former drinkers, and current drinkers of various levels. Incident diabetes was determined by blood glucose measurements and self-report. After adjustment for potential confounders, an increased risk of diabetes was found in men who drank >21 drinks/week when compared with men who drank < or =1 drink/week (odds ratio = 1.50, 95% confidence interval: 1.02, 2.20) while no significant association was found in women. This increased diabetes risk among men who drank >21 drinks/week was predominantly related to spirits rather than to beer or wine consumption. The relative odds of incident diabetes in a comparison of men who drank >14 drinks of spirits per week with men who were current drinkers but reported no regular use of spirits, beer, or wine were 1.82 (95% confidence interval: 1.14, 2.92). Results of this study support the hypothesis that high alcohol intake increases diabetes risk among middle-aged men. However, more moderate levels of alcohol consumption do not increase risk of type 2 diabetes in either middle-aged men or women.
Accumulating evidence suggests that the cytosolic calcium-independent phospholipase A(2) (iPLA(2)beta) manifests a signaling role in insulin-secreting (INS-1) beta-cells. Earlier, we reported that insulin-secretory responses to cAMP-elevating agents are amplified in iPLA(2)beta-overexpressing INS-1 cells (Ma Z, Ramanadham S, Bohrer A, Wohltmann M, Zhang S, and Turk J. J Biol Chem 276: 13198-13208, 2001). Here, immunofluorescence, immunoaffinity, and enzymatic activity analyses are used to examine distribution of iPLA(2)beta in stimulated INS-1 cells in greater detail. Overexpression of iPLA(2)beta in INS-1 cells leads to increased accumulation of iPLA(2)beta in the nuclear fraction. Increasing glucose concentrations alone results in modest increases in insulin secretion, relative to parental cells, and in nuclear accumulation of the iPLA(2)beta protein. In contrast, cAMP-elevating agents induce robust increases in insulin secretion and in time-dependent nuclear accumulation of iPLA(2)beta fluorescence, which is reflected by increases in nuclear iPLA(2)beta protein content and specific enzymatic activity. The stimulated effects are significantly attenuated in the presence of cell-permeable inhibitors of protein phosphorylation and glycosylation. These findings suggest that conditions that amplify insulin secretion promote translocation of beta-cell iPLA(2)beta to the nuclei, where it may serve a crucial signaling role.
Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane. The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes. The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae. We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism. We demonstrate that prohibitins in C. elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans. By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad. We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells. This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.
Obesity is an increasingly prevalent human condition in developed societies. Despite major progress in the understanding of the molecular mechanisms leading to obesity, no safe and effective treatment has yet been found. Here, we report an antiobesity therapy based on targeted induction of apoptosis in the vasculature of adipose tissue. We used in vivo phage display to isolate a peptide motif (sequence CKGGRAKDC) that homes to white fat vasculature. We show that the CKGGRAKDC peptide associates with prohibitin, a multifunctional membrane protein, and establish prohibitin as a vascular marker of adipose tissue. Targeting a proapoptotic peptide to prohibitin in the adipose vasculature caused ablation of white fat. Resorption of established white adipose tissue and normalization of metabolism resulted in rapid obesity reversal without detectable adverse effects. Because prohibitin is also expressed in blood vessels of human white fat, this work may lead to the development of targeted drugs for treatment of obese patients.
Overexpression of adenine nucleotide translocase-1 (ANT1) is known to induce apoptosis (Bauer, M. K., Schubert, A., Rocks,
O., and Grimm, S. (1999) J. Cell Biol. 147, 1493–1501), but the mechanisms involved remain unclear. In this study we show that ANT1 overexpression results in a
recruitment of the IκBα-NF-κB complex into mitochondria, with a coincident decrease in nuclear NF-κB DNA binding activity.
In this situation, NF-κB transcriptionally regulated genes with antiapoptotic activity, such as Bcl-XL, MnSOD2, and c-IAP2, are down-regulated, and consequently, cells are sensitized to apoptosis. Accordingly, co-expression of p65 partially interferes
with the proapoptotic effect of ANT1 overexpression. Despite the high identity of the two isoforms, overexpression of ANT2
does not exert an apoptotic effect; this lack of apoptotic activity is correlated with the absence of mitochondrial IκBα-NF-κB
recruitment or changes in NF-κB activity. Thus, we propose that the mitochondrial recruitment of NF-κB observed following
ANT1 overexpression has an important role in ANT1 proapoptotic activity.
N-(2-hydroxyethyl)-nicotinamide nitrate (nicorandil) is a unique anti-anginal agent, reported to act as both an ATP-sensitive K(+) channel opener (PCO) and a nitric oxide donor. It also has an anti-oxidant action. We examined the effects of nicorandil on streptozotocin (STZ)-induced islet beta-cell damage both in vivo and in vitro.
STZ-induced diabetic Brown Norway rats (STZ-DM) were fed with nicorandil-containing chow from day 2 (STZ-DM-N48), 3 (STZ-DM-N72), and 4 (STZ-DM-N96) to day 30. Body weight, blood glucose, and plasma insulin were measured every week. For the in vitro assay, neonatal rat islet-rich cultures were performed and cells were treated with nicorandil from 1 h before to 2 h after exposure to STZ for 30 min. Insulin secretion from islet cells was assayed after an additional 24 h of culture. We also observed the effect of nicorandil on the generation of reactive oxygen species (ROS) from rat inslinoma cells (RINm5F).
Body weight loss and blood glucose levels of STZ-DM-N48 rats were significantly lower than those of STZ-DM rats. Immunohistochemical staining of insulin showed preservation of insulin-secreting islet beta-cells in STZ-DM-N48 rats. Nicorandil also dose-dependently recovered the insulin release from neonatal rat islet cells treated with STZ in in vitro experiments. Nicorandil did not act as a PCO on neonatal rat islet beta-cells or RINm5F cells, and did not show an inhibitory effect on poly(ADP-ribose) polymerase-1. However, the drug inhibited the production of ROS stimulated by high glucose (22.0 mmol/l) in RINm5F cells.
These results suggested that nicorandil improves diabetes and rat islet beta-cell damage induced by STZ in vivo and in vitro. It protects islet beta-cells, at least partly, via a radical scavenging effect.
Adipocytes hold the body's major energy reserve as triacylglycerols packaged in large lipid droplets. Perilipins, the most abundant proteins on these lipid droplets, play a critical role in facilitating both triacylglycerol storage and hydrolysis. The stimulation of lipolysis by beta-adrenergic agonists triggers rapid phosphorylation of perilipin and translocation of hormone-sensitive lipase to the surfaces of lipid droplets and more gradual fragmentation and dispersion of micro-lipid droplets. Because few lipid droplet-associated proteins have been identified in adipocytes, we isolated lipid droplets from basal and lipolytically stimulated 3T3-L1 adipocytes and identified the component proteins by mass spectrometry. Structural proteins identified in both preparations include perilipin, S3-12, vimentin, and TIP47; in contrast, adipophilin, caveolin-1, and tubulin selectively localized to droplets in lipolytically stimulated cells. Lipid metabolic enzymes identified in both preparations include hormone-sensitive lipase, lanosterol synthase, NAD(P)-dependent steroid dehydrogenase-like protein, acyl-CoA synthetase, long chain family member (ACSL) 1, and CGI-58. 17-beta-Hydroxysteroid dehydrogenase, type 7, was identified only in basal preparations, whereas ACSL3 and 4 and two short-chain reductase/dehydrogenases were identified on droplets from lipolytically stimulated cells. Additionally, both preparations contained FSP27, ribophorin I, EHD2, diaphorase I, and ancient ubiquitous protein. Basal preparations contained CGI-49, whereas lipid droplets from lipolytically stimulated cells contained several Rab GTPases and tumor protein D54. A close association of mitochondria with lipid droplets was suggested by the identification of pyruvate carboxylase, prohibitin, and a subunit of ATP synthase in the preparations. Thus, adipocyte lipid droplets contain specific structural proteins as well as lipid metabolic enzymes; the structural reorganization of lipid droplets in response to the hormonal stimulation of lipolysis is accompanied by increases in the relative mass of several proteins and the recruitment of additional proteins.
Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of
adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to
profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation
was screened, and 41 proteins that responded to blocking of secretion by 20°C treatment and/or brefeldin A treatment
were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as
secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25
are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an
actively remodeling extracellular matrix and several factors involved in growth regulation.
The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing beta-cell probably equals that of ATP production. In glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high and correlates more strongly with the glucose concentration the beta-cell is exposed to (and thus with insulin release) than does pyruvate decarboxylation, which produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The carboxylation pathway can increase the levels of citric acid cycle intermediates, and this indicates that anaplerosis, the net synthesis of cycle intermediates, is important for insulin secretion. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates have roles in signaling insulin secretion. Although evidence is quite good that all physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric acid cycle intermediates. This article speculates on their potential roles as signaling molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and malonyl-CoA, as well as alpha-ketoglutarate as a substrate for hydroxylases. We also discuss the "succinate mechanism," which hypothesizes that insulin secretagogues produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria in causing oscillations in beta-cell citrate levels. These parallel oscillations in ATP and NAD(P)H. Oscillations in beta-cell plasma membrane electrical potential, ATP/ADP and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with pulsatile insulin release. Citrate oscillations might synchronize oscillations of individual mitochondria with one another and mitochondrial oscillations with oscillations in glycolysis and, therefore, with flux of pyruvate into mitochondria. Thus citrate oscillations may synchronize mitochondrial ATP production and anaplerosis with other cellular oscillations.
The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.
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.
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.
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.
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.
Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits alpha-1 and beta-2, vimentin, gamma-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.
Evidence regarding the association between alcohol consumption and type 2 diabetes risk remains inconsistent, particularly with regard to male-female differences. The authors conducted a prospective study of type 2 diabetes risk associated with alcohol consumption in a cohort of 12,261 middle-aged participants of the Atherosclerosis Risk in Communities Study (1990-1998), who were followed between 3 and 6 years. Alcohol consumption at baseline was characterized into lifetime abstainers, former drinkers, and current drinkers of various levels. Incident diabetes was determined by blood glucose measurements and self-report. After adjustment for potential confounders, an increased risk of diabetes was found in men who drank >21 drinks/week when compared with men who drank 21 drinks/week was predominantly related to spirits rather than to beer or wine consumption. The relative odds of incident diabetes in a comparison of men who drank >14 drinks of spirits per week with men who were current drinkers but reported no regular use of spirits, beer, or wine were 1.82 (95% confidence interval: 1.14, 2.92). Results of this study support the hypothesis that high alcohol intake increases diabetes risk among middle-aged men. However, more moderate levels of alcohol consumption do not increase risk of type 2 diabetes in either middle-aged men or women. Am J Epidemiol 2001;154:748-57.
Five cosmid clones, isolated by procedures to screen genomic libraries for homologous variants of the human prohibitin gene (PHB), were analyzed to determine their genomic structures. Four of these (PHBP1-4) were found to be processed pseudogenes, each located on a different chromosome from their counterparts on chromosome 17q21. The DNA sequence of one clone (PHBP1, on chromosome 6q25) shared a 91.3% identity at the nucleotide level with the cDNA of functional prohibitin. A large number of human tumors of the breast, ovary, liver, and lung were examined for somatic mutations in the PHB gene. Although mutations were observed in a few sporadic breast cancers, none were identified in any of the other cancers.
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.
Prohibitin (PHB), a protein located on the inner mitochondrial membrane and nuclei, is an intracellular effector of transforming growth factor-beta (TGF-beta) signaling in prostate cancer cells. This study investigated the involvement of PHB in the apoptosis and survival outcomes of human prostate cancer cell to TGF-beta. shRNA PHB loss of function in prostate cancer cells led to enhanced apoptotic response to TGF-beta via Smad-dependent mechanism.
TGF-beta activation of Raf-Erk intracellular signaling, led to PHB phosphorylation, decreased inner mitochondrial permeability, and increased cell survival. Calcein-based immunofluorescence studies revealed the functional involvement of PHB in maintaining inner mitochondrial membrane permeability as an integral component of TGF-beta induced apoptosis in prostate cancer cells.
These finding indicates that induction of TGF-beta apoptosis is mediated by Smad-dependent and Smad-independent signaling (MAPK) converging at PHB as a downstream effector regulating inner mitochondrial permeability. Putative PHB associated proteins were identified by subjecting TGF-beta treated cells to immunoprecipitation with anti-PHB, and mass spectrometry. A screen for the kinase specific phosphorylation sites of PHB revealed three protein kinase (PKC) binding sites.
Our results demonstrate that TGF-beta led to upregulation of the PKC inhibitor 14-3-3 protein and promoted its association with PHB, while PHB association with PKC-delta, was inhibited by the MEK1 inhibitor, documenting a critical interdependence between the MEK-ERK signaling and prohibitin phosphorylation. These findings suggest a dual role for PHB as a downstream determinant of the cellular response to TGF-beta via Smad-dependent pathway (apoptosis) and MAPK intracellular signaling (survival).
Apically expressed human MUC1 is known to become endocytosed and either to re-enter the secretory pathway for recycling to the plasma membrane or to be exported by the cells via the formation of multi-vesicular bodies and the release of exosomes. By using recombinant fusion-tagged MUC1 as a bait protein we followed an anti-myc affinity-based approach for isolating subpopulations of lipid rafts from the plasma membranes and exosomes of MCF-7 breast cancer cells. MUC1(+) lipid rafts were not only found to contain genuine raft proteins (flotillin-1, prohibitin, G protein, annexin A2), but also raft-associated proteins linking these to the cytoskeleton (ezrin/villin-2, profilin II, HSP27, gamma-actin, beta-actin) or proteins in complexes with raft proteins, including the bait protein (HSP60, HSP70). Major overlaps were revealed for the subproteomes of plasma membranous and exosomal lipid raft preparations, indicating that MUC1 is sorted into subpopulations of rafts for its trafficking via flotillin-dependent pathways and export via exosomes.
Effects of ethanol and its metabolites, acetaldehyde and acetate, on insulin secretion were studied in isolated islets from normal rats. Addition of ethanol to the incubation media inhibited glucose mediated insulin release in a dose related manner. Prior exposure of islets to ethanol during the preincubation period had no influence on subsequent insulin response to either glucose or glucose plus ethanol. Acetaldehyde inhibited while sodium acetate potentiated insulin response to glucose. It is concluded that ethanol has a direct inhibitory effect on glucose mediated insulin release from rat islets.
This study was done to delineate the role of alpha- and beta-adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an alpha-adrenergic blocker, phentolamine, or a beta-adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.
In vivo and in vitro effects of ethanol on the kinetics of insulin secretion in response to glucose and tolbutamide were studied in perifused rat islets. Phases I and II insulin response to 16.7 m m glucose was decreased 46% and 48%, respectively, in islets of rats given ethanol intragastrically 1 g/kg 1 h prior to sacrifice. Mean blood ethanol levels at the time of animal sacrifice were 19.4 mmol/l. The magnitude of insulin suppression was not significantly enhanced with higher ethanol doses, 2 or 3 g/kg, although mean blood ethanol levels increased to 25.9 and 60.3 mmol/l, respectively. Similarly, significant inhibition of both phases of insulin response to glucose occurred when ethanol 1 or 3 g/kg was given intraperitoneally instead of orally. Ethanol had no effect on insulin secretion when given orally 4 h instead of 1 h prior to islet isolation.
Ethanol, 65 mmol/l, added directly to rat islets perifusate simultaneously with 16.7 m m glucose decreased both phases I and II insulin response nearly half; whereas addition of 21.7 instead of 65 mmol/l ethanol had no effect. Pre-treatment of islets with 21.7 or 65 mmol/l ethanol during 30 min basal islets perifusion period had no effect on subsequent insulin response to 16.7 m m glucose. Insulin response to 10 m m tolbutamide was decreased nearly 81% by the simultaneous presence of 65 mmol/l ethanol in islets perifusate.
The effects of varying concentrations of ethanol (1,10, and 30 mM) and its metabolites (1 mM acetate and 1 and 10 mM acetaldehyde) on insulin and glucagon secretion induced by glucose (11.1 mM) and arginine (20 mM) were studied in isolated perfused pancreas of Sprague-Dawley rats
Ethanol and its metabolites did not significantly modify basal secretion of the two hormones. Ethanol reduced glucose-induced insulin secretion by means of a dose-related effect. Arginine-induced insulin output did not seem to be influenced to any significant degree. Acetate and acetaldehyde significantly inhibited glucose and arginine-induced insulin secretion. While ethanol (10 and 30 mM) did not modify glucagon output during arginine perfusion, acetate and acetaldehyde markedly enhanced it.
The block of insulin secretion and the increased secretion of glucagon could explain the diabetogenic effect of ethanol demonstrated in vivo. The mechanism by which ethanol acts on the pancreatic β- and α-cells is discussed.
During the course of two years, 76 representative subjects seen in a community hospital emergency room who admitted to having recently used alcohol while still appearing sober had their blood alcohol levels measured to determine the levels of blood alcohol present in ambulatory sober alcohol users. As a group the mean blood alcohol level obtained in those who had measurable levels was 268 ± 10 mg/dl mean ± SEM). More men (47) than women (18) admitted to having used ethanol and had measurable blood ethanol levels and therefore were studied. Moreover, the mean blood alcohol level in the men studied was arithmetically greater (272 ± 13 mg/d1) than that present in the women (260 ± 13mg/d1). The range of alcohol levels seen in the two sexes, however, were quite similar. Using a blood alcohol level > 200 mg/dl in a clinically “non-intoxicated” individual as the cut-off level for defining one as a suspect chronic alcohol user, our data would suggest that such individuals not uncommonly have blood alcohol levels as high as 290 ± 9 mg/dl.
Chronic ethanol exposure inhibits the capacity of the liver to regenerate. Insulin is a potent hepatotrophic factor and it was determined if ethanol interferes with insulin receptor substrate (IRS-1)-protein mediated signal transduction during liver regeneration. Tyrosyl phosphorylation of IRS-1 was strikingly increased prior to the major wave of DNA synthesis in isocaloric pair-fed control rats; a blunted and delayed response was found in ethanol-fed rats. Enzymatic activity of phosphatidylinositol 3-kinase, a Src homology 2 (SH2) domain containing signal transduction molecule was enhanced by the association with tyrosyl phosphorylated IRS-1, whereas in ethanol-fed rats, this activity was greatly diminished and delayed. These results indicate that one potential molecular mechanism whereby ethanol inhibits hepatocyte DNA synthesis is through its action on the IRS-1-mediated signal transduction cascade.
Quantitative analysis of the light microscopic and fine structure of rat islet B-cells was carried out in chronic alcoholism. Absolute pancreatic weight and volume were similar in groups C (control) and E (ethanol), but relative pancreatic weight in group E rat was decreased. The results for fasting blood glucose and insulin levels were similar in the two groups of animals. There was a significantly reduced total pancreatic islet volume in E rats. The total number of endocrine cells both per islet and per microns2 of islet was similar in the two groups of animals. The volume density and number of B-cells per islet and per microns2 of islet were not changed in ethanol-treated rats as compared with the control. On the other hand, diameter, surface area and volume of the B-cells and their nuclei were found to be statistically significantly decreased. Histological examination revealed that islet blood vessels were dilated in alcoholic rats. Over the 4-month period of ethanol intake a significant decrease in cell profile area, nuclear profile area and volume density of cytoplasmic granules and an increase in the profile area and volume density of endoplasmic reticulum occurred. The gross histological alteration seen in most B-cells of the ethanol-treated rats was irregularity of the nuclear envelope with deep invagination and with margination of heterochromatin and many empty granules or granules without clear electron dense crystals of insulin. The present results indicate some optical and structural abnormalities of B-cells in chronic alcoholism that may be related to cell dysfunction and may contribute, at least in part, to the endocrine pancreas functional disturbance.
Five cosmid clones, isolated by procedures to screen genomic libraries for homologous variants of the human prohibitin gene (PHB), were analyzed to determine their genomic structures. Four of these (PHBP1-4) were found to be processed pseudogenes, each located on a different chromosome from their counterparts on chromosome 17q21. The DNA sequence of one clone (PHBP1, on chromosome 6q25) shared a 91.3% identity at the nucleotide level with the cDNA of functional prohibitin. A large number of human tumors of the breast, ovary, liver, and lung were examined for somatic mutations in the PHB gene. Although mutations were observed in a few sporadic breast cancers, none were identified in any of the other cancers.
Cellular senescence is determined by multiple factors, including the genetic regulation of metabolism and responses to endogenous and exogenous stresses [1-4]. Recent studies implicate a limited number of gene products in elongating lifespan in yeast and Caenorhabditis elegans [2-4]; these include the C, elegans gene cik-1, a central regulator of metabolism [5], and yeast RAS2, which controls the response to ultraviolet irradiation and other stresses [3]. Another gene postulated to effect senescence is PHB1, the yeast homologue of prohibitin [3], a rodent gene initially identified as a potential regulator of growth arrest and tumour suppressor [6-8]. Highly conserved prohibitin homologues have been identified in mammals [9], Drosophila [10], C. elegans [9], plants [11] and yeast. A second mammalian gene, encoding BAP37, a protein with sequence similarity to prohibitin, is thought to be involved in lymphocyte function [9]. Here, we show that the nuclear-encoded mammalian prohibitin and BAP37 proteins are present in mitochondria, are co-expressed, and interact physically with each other. Deletion of the Saccharomyces cerevisiae homologues, PHB1 and PHB2, results in a decreased replicative lifespan and a defect in mitochondrial membrane potential. Our observations highlight the relationship between the metabolic efficiency of cells and the ageing process, and provide evidence for its evolutionary conservation.
Ethanol impairs insulin-stimulated survival and mitochondrial function in immature proliferating neuronal cells due to marked inhibition of downstream signaling through P13 kinase. The present study demonstrates that, in contrast to immature neuronal cells, the major adverse effect of chronic ethanol exposure (50 mM) in post-mitotic rat cerebellar granule neurons is to inhibit insulin-stimulated mitochondrial function (MTT activity, MitoTracker Red fluorescence, and cytochrome oxidase immunoreactivity). Ethanol-impaired mitochondrial function was associated with increased expression of the p53 and CD95 pro-apoptosis genes, reduced Calcein AM retention (a measure of membrane integrity), increased SYTOX Green and propidium iodide uptake (indices of membrane permeability), and increased oxidant production (dihydrorosamine fluorescence and H2O2 generation). The findings of reduced membrane integrity and mitochondrial function in short-term (24 h) ethanol-exposed neurons indicate that these adverse effects of ethanol can develop rapidly and do not require chronic neurotoxic injury. A role for caspase activation as a mediator of impaired mitochondrial function was demonstrated by the partial rescue observed in cells that were pre-treated with broad-spectrum caspase inhibitors. Finally, we obtained evidence that the inhibitory effects of ethanol on mitochondrial function and membrane integrity were greater in insulin-stimulated compared with nerve growth factor-stimulated cultures. These observations suggest that activation of insulin-independent signaling pathways, or the use of insulin sensitizer agents that enhance insulin signaling may help preserve viability and function in neurons injured by gestational exposure to ethanol.
Chronic gestational exposure to ethanol has profound adverse effects on brain development. In this regard, studies using in vitro models of ethanol exposure demonstrated impaired insulin signaling mechanisms associated with increased apoptosis and reduced mitochondrial function in neuronal cells. To determine the relevance of these findings to fetal alcohol syndrome, we examined mechanisms of insulin-stimulated neuronal survival and mitochondrial function using a rat model of chronic gestational exposure to ethanol. In ethanol-exposed pups, the cerebellar hemispheres were hypoplastic and exhibited increased apoptosis. Isolated cerebellar neurons were cultured to selectively evaluate insulin responsiveness. Gestational exposure to ethanol inhibited insulin-stimulated neuronal viability, mitochondrial function, Calcein AM retention (membrane integrity), and GAPDH expression, and increased dihydrorosamine fluorescence (oxidative stress) and pro-apoptosis gene expression (p53, Fas-receptor, and Fas-ligand). In addition, neuronal cultures generated from ethanol-exposed pups had reduced levels of insulin-stimulated Akt, GSK-3beta, and BAD phosphorylation, and increased levels of non-phosphorylated (activated) GSK-3beta and BAD protein expression. The aggregate results suggest that insulin-stimulated central nervous system neuronal survival mechanisms are significantly impaired by chronic gestational exposure to ethanol, and that the abnormalities in insulin signaling mechanisms persist in the early postnatal period, which is critical for brain development.
Various epidemiological studies suggest that alcohol intake is one of the risk factors leading to type II or non-insulin-dependent diabetes mellitus (NIDDM), but the effect of alcohol on beta-cell function remains unexplored. To study the mechanism of the diabetogenic action of ethanol, we investigated the effect of ethanol on beta-cell functions using a single clonal beta-cell line, HIT-T15 cells. When HIT cells were treated with ethanol, the metabolic activity judged by MTT assay was inhibited in dose- and time dependent manners, but cytotoxicity was not observed. Ethanol also inhibited basal insulin secretion by 30% compared to the untreated control. However, glucose-stimulated insulin secretion was not impaired by ethanol although the basal insulin secretion was inhibited. These results imply that ethanol exert beta-cells to overwork in order to compensate inhibition of the basal secretion. This finding may at least in part explain the diabetogenic action of ethanol.
This article has no abstract; the first 100 words appear below.
A number of epidemiologic studies have found an association of alcohol intake with a reduced risk of cardiovascular disease. These observations have been purported to explain the so-called French paradox: the lower rate of cardiovascular disease in France than in its neighboring countries to the north and in the United States, despite the relatively high intake of fat in France. Several studies attribute the French paradox to the relatively high consumption of red wine by the French.¹ Other epidemiologic studies,²,³ including that of Mukamal et al. in this issue of the Journal, ⁴ suggest that all alcoholic beverages, not wine . . .
Source Information
From the Division of Preventive Medicine and Nutrition, Columbia University College of Physicians and Surgeons, New York.
High ethanol intake is considered to impair insulin sensitivity. In the present study, we investigated the acute and chronic effects of ethanol intake on glucose metabolism and insulin signal transduction. Hyperinsulinemic-euglycemic clamp studies revealed 70% and 51% decreases in the glucose infusion rate, 52% and 31% decreases in the glucose utilization rate, and 6.6- and 8.0-fold increases in hepatic glucose in continuous- and acute-ethanol-loaded rats, respectively. Despite the presence of insulin resistance, alcohol-fed rats showed enhanced tyrosine phosphorylation of insulin receptors, IRS-1 and IRS-2, induced by insulin injection via the portal vein. PI 3-kinase activities associated with IRSs and phosphotyrosine also increased significantly as compared with those of controls. These data suggest ethanol intake to be a factor leading to insulin resistance, regardless of whether it is a single or continuous intake. In addition, the insulin signaling step impaired by ethanol feeding is likely to be downstream from PI 3-kinase.
Alcohol is a constituent of the diet that is generally taken in on a voluntary basis. The amount and type of alcohol consumed along with the frequency of alcohol consumption can vary tremendously and can have divergent effects on an organism. Animal models have been developed to investigate the mechanisms by which both acute alcohol administration and chronic alcohol consumption affect the various organ systems of the body. The deleterious effects of alcohol, at least partly involve alcohol induced oxidative injury that has been documented by measurement of oxidant radicals, alterations in oxidant/antioxidant balance and oxidant induced changes in cellular proteins and lipids. In addition, evidence for alcohol-induced oxidant injury comes from studies in which pretreatment with antioxidants such as vitamin E, vitamin C, and agents that enhance antioxidant capacity attenuate alcohol induced effects. The susceptibility of tissues to alcohol-induced injury is related to their function and the method by which they are exposed to alcohol. For example, the stomach and liver are exposed to the highest concentrations upon ingestion and absorption of alcohol. The liver is also the major organ for metabolism, and with chronic alcohol use, P450 2E1 is induced. This enzyme activity however, adds additional oxidative stress to the liver. Although antioxidants can attenuate alcohol-induced injury, there is no one antioxidant that protects all organs during all modes of exposure. In addition, more studies are needed to determine if administration of antioxidants after alcohol exposure can reverse alcohol induce tissue damage. This review will summarize results from experiments in which alcohol has been delivered for a short time (acute) or prolonged period (chronic); in vivo or in vitro; at physiologic doses or at supraphysiologic doses. The effects of alcohol on various tissues will be presented and finally, the contribution of oxidant injury to alcohol induced tissue damage will be discussed.
Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity. The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function. Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane. Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast. With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected. This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain. It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast. Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains. Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter. They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell.
Early diagnosis of cancer is crucial for successful treatment. Noninvasive assays for detecting tumor-derived antigens in serum and other bodily fluids have the potential to screen healthy individuals for hitherto undetected cancers. Very few such assays have been successfully developed, in part because identifying potential target antigens remains a challenge. To identify new blood-borne tumor antigens for the purpose of establishing such assays, we have developed a novel technique called differential immunization. Using this method, we have identified PHB1 and PHB2, proteins thought to function as mitochondrial chaperones and transcriptional regulators, as antigens released from colorectal tumors in vivo. Serum from colorectal patients contains significantly higher levels of these antigens compared to serum from healthy volunteers. These data demonstrate that differential immunization is an effective new method for identifying tumor-derived antigens in serum.
Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen species (ROS) and mitochondrial dysfunction. Treatment with antioxidants can significantly reduce the degree of damage. Despite well established roles of ROS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS are poorly characterized. We hypothesized that certain cysteinyl residues of target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. A targeted proteomics approach utilizing biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized cysteinyl residues was employed to investigate which mitochondrial proteins are modified during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 100 mM ethanol for 4 and 8 h, the biotin-NM-labeled oxidized proteins were purified with agarose coupled to either streptavidin or monoclonal antibody against biotin. The purified proteins were resolved by two-dimensional gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and analyzed for identity utilizing either matrix-assisted laser desorption-time of flight mass spectrometry or microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. The results demonstrate that heat shock protein 60, protein disulfide isomerase, mitochondrial aldehyde dehydrogenases, prohibitin, and other proteins were oxidized after alcohol exposure. The identity of some of the proteins purified with streptavidin-agarose was also confirmed by immunoblot analyses using the specific antibody to each target protein. This method was also used to identify oxidized mitochondrial proteins in the alcohol-fed mouse liver. These results suggest that exposure to ethanol causes oxidation of various mitochondrial proteins that may negatively affect their function and contribute to alcohol-induced mitochondrial dysfunction and cellular injury.
Vi capsular polysaccharide (Vi) was first identified as a virulence antigen of Salmonella typhi, the causative agent of typhoid fever in humans; it renders S. typhi resistant to phagocytosis and the action of serum complement. However, the role of Vi during the infection of intestinal epithelium with S. typhi is not completely understood. We show here that Vi can interact with a model human intestinal epithelial cell line, Caco-2, through a cell-surface-associated molecular complex containing two major proteins of 30 and 35 kDa and a minor protein of ≈68 kDa. The two major proteins were identified as the putative tumor suppressor molecule, prohibitin, and its closely related homolog, B cell receptor-associated protein 37. These two proteins were enriched in lipid rafts, and Vi readily associated with these membrane microdomains. Engagement of Caco-2 cells with Vi inhibited their ability to produce an inflammatory response upon infection with Vi–S. typhi. Consistent with this effect, infection of Caco-2 cells with Vi⁺S. typhi produced less IL-8 compared with Vi–S. typhi. Cells treated with Vi showed reduced extracellular signal-regulated kinase phosphorylation in response to infection with Vi–S. typhi or stimulation with phorbol 12-myristate 13-acetate, suggesting that the mitogen-activated protein kinase pathway might be a target for Vi-mediated inhibition of inflammatory responses. These findings reveal a crucial role for Vi in the modulation of early inflammatory responses during infection with S. typhi. This kind of a modulation could play a significant role in the establishment of infection by S. typhi.
• Vi capsular polysaccharide
• putative tumor suppressor molecule
• IL-8
Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function and protection against senescence. There is considerable controversy concerning the function of nuclear-localized PHB. PHB has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. PHB might also function as a cell-surface receptor for an as-yet unidentified ligand. Cell-associated PHB in the gastrointestinal tract has been implicated in protection against infection and inflammation and the induction of apoptosis in other tissues. The diverse array of functions of PHB, together with the emerging evidence that its function can be modulated specifically in certain tissues, suggest that targeting PHB would be a useful therapeutic approach for the treatment of variety of disease states, including inflammation, obesity and cancer.