Association of copy number variation in X chromosome-linked PNPLA4 with heterotaxy and congenital heart disease.

BACKGROUND: Heterotaxy (HTX) is a thoracoabdominal organ anomaly syndrome and commonly accompanied by congenital heart disease (CHD). The aim of this study was to analyze rare copy number variations (CNVs) in a HTX/CHD cohort and to examine the potential mechanisms contributing to HTX/CHD. METHODS: Chromosome microarray analysis was used to identify rare CNVs in a cohort of 120 unrelated HTX/CHD patients, and available samples from parents were used to confirm the inheritance pattern. Potential candidate genes in CNVs region were prioritized via the DECIPHER database, and PNPLA4 was identified as the leading candidate gene. To validate, we generated PNPLA4-overexpressing human induced pluripotent stem cell lines as well as pnpla4-overexpressing zebrafish model, followed by a series of transcriptomic, biochemical and cellular analyses. RESULTS: Seventeen rare CNVs were identified in 15 of the 120 HTX/CHD patients (12.5%). Xp22.31 duplication was one of the inherited CNVs identified in this HTX/CHD cohort, and PNPLA4 in the Xp22.31 was a candidate gene associated with HTX/CHD. PNPLA4 is expressed in the lateral plate mesoderm, which is known to be critical for left/right embryonic patterning as well as cardiomyocyte differentiation, and in the neural crest cell lineage. Through a series of in vivo and in vitro analyses at the molecular and cellular levels, we revealed that the biological function of PNPLA4 is importantly involved in the primary cilia formation and function via its regulation of energy metabolism and mitochondria-mediated ATP production. CONCLUSIONS: Our findings demonstrated a significant association between CNVs and HTX/CHD. Our data strongly suggested that an increased genetic dose of PNPLA4 due to Xp22.31 duplication is a disease-causing risk factor for HTX/CHD.

The Impact of the Fluid-Solid Coupling Behavior of Macro and Microstructures in the Spiral Cochlea on Hearing.

The cilia of the outer hair cells (OHCs) are the key microstructures involved in cochlear acoustic function, and their interactions with lymph in the cochlea involve complex, highly nonlinear, coupled motion and energy conversions, including macroscopic fluid-solid coupling. Recent optical measurements have shown that the frequency selectivity of the cochlea at high sound levels is entirely mechanical and is determined by the interactions of the hair bundles with the surrounding fluid. In this paper, an analytical mathematical model of the spiral cochlea containing macro- and micromeasurements was developed to investigate how the phonosensitive function of OHCs' motions is influenced by the macrostructural and microstructural fluid-solid coupling in the spiral cochlea. The results showed that the macrostructural and microstructural fluid-solid coupling exerted the radial forces of OHCs through the flow field, deflecting the cilia and generating frequency-selective properties of the microstructures. This finding showed that microstructural frequency selectivity arises from the radial motions of stereocilia hair bundles and enhances the hearing of sound signals at specific frequencies. It also implied that the macrostructural and microstructural fluid-solid couplings influence the OHCs' radial forces and that this is a key factor in the excitation of ion channels that enables their activity in helping the brain to detect sound.

Effect of portulaca (Portulaca oleracea L.) extract on the quality and physicochemical attributes of vacuum-packed seasoned steaks during chilled storage.

BACKGROUND: Vacuum packaging has the ability to reduce oxidative deterioration and microbial-induced spoilage of meat. However, in an oxygen-free environment, it can lead to the development of an unappealing purplish-red color and a decrease in the water-holding capacity of meat, thereby impacting the overall meat quality. Portulaca oleracea L. (POL) is a homology of medicine and food known for its exceptional antioxidant and antimicrobial properties. RESULTS: The aim of our present study was to investigate the antioxidant and antimicrobial ability of n-butanol phase extract of POL and the effect of POL extract incorporation on the quality (water-holding capacity, shear force, color, and texture) and physicochemical (pH, total volatile base nitrogen, and total viable counts) attributes of vacuum-packed seasoned steaks at 4 °C over a 15-day period. Results showed that the POL extract had excellent antioxidant and antimicrobial capacity. Furthermore, the addition of POL extract significantly inhibited protein oxidation and microbial growth of steaks (P < 0.05), and improved the water-holding capacity, color properties, and tenderness (P < 0.05). Moreover, there were no significant differences (P > 0.05) in the color, water-holding capacity, or tenderness between the 0.5 and 1 g kg-1 POL extract treatment groups compared to the sodium nitrite control group. CONCLUSION: These results indicate that POL extract had the potential to replace sodium nitrite due to its ability to hinder protein oxidation and microbial growth of vacuum-packed seasoned steaks, while enhancing the color, water-holding capacity, and tenderness of seasoned steaks. © 2024 Society of Chemical Industry.

Changes in the Quality of Myofibrillar Protein Gel Damaged by High Doses of Epigallocatechin-3-Gallate as Affected by the Addition of Amylopectin.

This work investigated the improvement of amylopectin addition on the quality of myofibrillar proteins (MP) gel damaged by high doses of epigallocatechin-3-gallate (EGCG, 80 µM/g protein). The results found that the addition of amylopectin partially alleviated the unfolding of MP induced by oxidation and EGCG, and enhanced the structural stability of MP. Amylopectin blocked the loss of the free amine group and thiol group, and increased the solubility of MP from 7.0% to 9.5%. The carbonyl analysis demonstrated that amylopectin addition did not weaken the antioxidative capacity of EGCG. It was worth noting that amylopectin significantly improved the gel properties of MP treated with a high dose of EGCG. The cooking loss was reduced from 51.2% to 35.5%, and the gel strength was reduced from 0.41 N to 0.29 N after adding high concentrations of amylopectin (A:E(8:1)). This was due to that amylopectin filled the network of MP gel after absorbing water and changed into a swelling state, and partially reduced interactions between EGCG and oxidized MP. This study indicated that amylopectin could be used to increase the polyphenol loads to provide a more lasting antioxidant effect for meat products and improve the deterioration of gel quality caused by oxidation and high doses of EGCG.

Copy number variant analysis for syndromic congenital heart disease in the Chinese population.

BACKGROUND: Syndromic congenital heart disease (CHD) is among the most severe conditions in the pediatric population. Copy number variant (CNV) is an important cause of syndromic CHD, but few studies focused on CNVs related to these patients in China. The present study aimed to identify pathogenic CNVs associated with syndromic CHD in the Chinese population. METHODS: A total of 109 sporadic patients with syndromic CHD were applied chromosomal microarray analysis (CMA). Phenotype spectrum of pathogenic or likely pathogenic CNVs was analyzed. CHD-related genes were prioritized from genes within pathogenic or likely pathogenic CNVs by VarElect, OVA, AMELIE, and ToppGene. RESULTS: Using CMA, we identified 43 candidate CNVs in 37/109 patients. After filtering CNVs present in the general population, 29 pathogenic/likely pathogenic CNVs in 24 patients were identified. The diagnostic yield of CMA for pathogenic/likely pathogenic CNVs was 23.1% (24/104), excluding 5 cases with aneuploidies or gross chromosomal aberrations. The overlapping analysis of CHD-related gene lists from different prioritization tools highlighted 16 CHD candidate genes. CONCLUSION: As the first study focused on CNVs in syndromic CHD from the Chinese population, this study reveals the importance of CMA in exploring the genetic etiology of syndromic CHD and expands our understanding of these complex diseases. The bioinformatic analysis of candidate genes suggests several CHD-related genes for further functional research.

Time-domain analysis of a three-dimensional numerical model of the human spiral cochlea at medium intensity.

For the processing and detection of speech and music, the human cochlea has an exquisite sensitivity and selectivity of frequency and a dynamic range. How the cochlea performs these remarkable functions has fascinated auditory scientists for decades. Because it is not possible to measure sound-induced vibrations within the cochlea in a living human being, mathematical modeling has played an important role in cochlear mechanics. For this study, a three-dimensional human cochlear model with a fluid‒structure coupling was constructed. Time-domain analysis was performed to calculate the displacement, velocity, and stress of the basilar membrane (BM) and osseous spiral lamina (OSL) at different times in response to a pure tone stimulus. The model reproduced the traveling-wave motion of the BM. The model also showed that the cochlea's spiral shape can induce asymmetrical mechanical behavior of the BM and cause cochlear fluid to move in a radial direction; this may contribute to human sound perception. The cochlea's spiral shape not only enhances a low-frequency vibration of the BM but also changes the maximization of the positions of vibration. Therefore, the spiral's characteristics play a key role in the cochlea's frequency selectivity for low-frequency sounds. And this suggests that the OSL can react to sound as quickly as the BM. Furthermore, the basal region of the BM tends to have more stress than its other regions, and this may explain the clinical observation that human sensorineural hearing loss often occurs at high frequencies.

A Non-coding HES1 Variant Predisposes Children to Congenital Heart Disease in Chinese Population.

Background: As a key component in the NOTCH signaling pathway, HES1 plays an important role in vertebrate heart development. Variants in the HES1 coding sequence are known to be associated with congenital heart disease (CHD). However, little is known about HES1 non-coding sequence variants and their association with the risk of developing CHD. Method and Results: We initially analyzed the non-coding sequence of the HES1 gene in 12 unrelated CHD families by direct sequencing and identified a previously unreported promoter region variant (NM_005524.4: c.-1279-1278 insAC, rs148941464) in the HES1 gene in four CHD families. The homozygous variant in patients was inherited from carrier parents with normal phenotypes, indicating a likely recessive genetic model. Given that the HES1 gene is predicted to be likely to exhibit haploinsufficiency (%HI: 11.44), we hypothesized that the HES1 homozygous variant is a genetic risk factor underlying CHD. We then carried out sequencing of this HES1 variant in 629 sporadic non-syndromic CHD cases and 696 healthy controls and performed association analysis. Interestingly, we observed a significant association of the homozygous HES1 promoter variant with CHD (18.92% of cases vs. 9.91% of controls; OR: 2.291, 95% CI: 1.637-3.207, p = 9.72 × 10-7). No significant association with CHD was observed for the HES1 promoter heterozygous variant (p > 0.05). However, association analysis tests of the HES1 homozygous variant with each subtype of CHD revealed that this homozygous variant was strongly associated with transposition of the great arteries (TGA) (OR: 3.726, 95% CI: 1.745-7.956, p = 0.0003). Moreover, the prevalence of HES1 homozygous variants in CHD patients with TGA (27.66%) was significantly higher than that in patients with other CHD subtypes or controls. Similar results were observed in a replication group of TGA (n = 64). Functional studies demonstrated that the homozygous variant in the HES1 promoter can disrupt its ability to bind RXRA, an inhibitory transcription factor, which results in abnormally high expression of the HES1 gene, indicating that this variant harbors gain-of-function effects. Conclusions: Our findings reveal that the non-coding homozygous variant in the HES1 promoter has a gain-of-function effect and is associated with an increased risk of CHD development, especially the severe TGA subtype.

A novel de novo PDE4D gene mutation identified in a Chinese patient with acrodysostosis.

Acrodysostosis is an extremely rare disorder at birth, that is, characterized by skeletal dysplasia with short stature and midfacial hypoplasia, which has been reported to be caused by PDE4D and PRKAR1A gene mutations. Here, a Chinese boy with acrodysostosis, ventricular septal defect, and pulmonary hypertension was recruited for our study, and his clinical and biochemical characteristics were analyzed. A novel de novo heterozygous missense mutation (NM_001104631: c.2030A>C, p.Tyr677Ser) of the PDE4D gene was detected by whole exome sequencing and confirmed by Sanger sequencing. The c.2030A>C (p.Tyr677Ser) variant was located in exon 15 of the PDE4D gene, predicted to be damaging by a functional prediction program and shown to be highly conserved among many species. Further functional analysis showed that the p.Tyr677Ser substitution changes the function of the PDE4D protein, affects its subcellular localization in transfected cells, increases PDE4 activity in the regulation of cAMP signaling and affects cell proliferation. Our study identified a novel de novo PDE4D mutation in acrodysostosis of Chinese origin that not only contributes a deeper appreciation of the phenotypic characteristics of patients with PDE4D mutations but also expands the spectrum of PDE4D mutations.

Functional analysis of rare variants of GATA4 identified in Chinese patients with congenital heart defect.

Congenital heart defect (CHD) is one of the most common cardiovascular diseases, affecting approximately 0.8% of live births. The transcription factor GATA4 has been known to play a key role in cardiac development. In this study, we performed whole exome sequencing in nine unrelated CHD patients and found two rare deleterious missense variants in the GATA4 gene (c.C487T,p.P163S and c.C1223A,p.P408Q) (ExAC <0.001 and CADD >15) in three cases that were confirmed by Sanger sequencing. Subsequently, these two variants were screened for in an additional 226 patients with CHD and 206 healthy controls by Sanger sequencing, and no variants were observed. These two variants were predicted to be damaging to protein function using a functional prediction program. Co-IP indicated that both of the GATA4 variants (P163S and P408Q) blocked heterodimer formation between GATA4 and ZFPM2 protein. Immunofluorescence showed that the two GATA4 variants diminished the colocalization formation between GATA4 and ZFPM2 protein compared to that of WT protein. These findings indicate that the two rare variants of GATA4 might disturb its interaction with ZFPM2 and influence corresponding downstream gene activity, suggesting that the GATA4 variants may be associated with the pathogenesis of CHD.

CC-401 Promotes ß-Cell Replication via Pleiotropic Consequences of DYRK1A/B Inhibition.

Pharmacologic expansion of endogenous ß cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control ß-cell growth we screened ∼2400 bioactive compounds for rat ß-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat ß-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) ß-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat ß cells, which were broadly growth responsive to compound treatment, human ß-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3ß (GSK-3ß) or activin A receptor type II-like kinase/transforming growth factor-ß (ALK5/TGF-ß) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human ß-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced ß-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the ß-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control ß-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control ß-cell growth.

Genetic Disruption of Adenosine Kinase in Mouse Pancreatic ß-Cells Protects Against High-Fat Diet-Induced Glucose Intolerance.

Islet ß-cells adapt to insulin resistance through increased insulin secretion and expansion. Type 2 diabetes typically occurs when prolonged insulin resistance exceeds the adaptive capacity of ß-cells. Our prior screening efforts led to the discovery that adenosine kinase (ADK) inhibitors stimulate ß-cell replication. Here, we evaluated whether ADK disruption in mouse ß-cells affects ß-cell mass and/or protects against high-fat diet (HFD)-induced glucose dysregulation. Mice targeted at the Adk locus were bred to Rip-Cre and Ins1-Cre/ERT1Lphi mice to enable constitutive (ßADKO) and conditional (ißADKO) disruption of ADK expression in ß-cells, respectively. Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion (GSIS) were longitudinally monitored in normal chow (NC)-fed and HFD-fed mice. In addition, ß-cell mass and replication were measured by immunofluorescence-based islet morphometry. NC-fed adult ßADKO and ißADKO mice displayed glucose tolerance, insulin tolerance and ß-cell mass comparable to control animals. By contrast, HFD-fed ßADKO and ißADKO animals had improved glucose tolerance and increased in vivo GSIS. Improved glucose handling was associated with increased ß-cell replication and mass. We conclude that ADK expression negatively regulates the adaptive ß-cell response to HFD challenge. Therefore, modulation of ADK activity is a potential strategy for enhancing the adaptive ß-cell response.

A High-content In Vitro Pancreatic Islet ß-cell Replication Discovery Platform.

Loss of insulin-producing ß-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet ß-cells remains an attractive strategy. ß-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain ß-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify ß-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a ß-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on ß-cells and the molecular mechanisms that regulate ß-cell growth.

Oral glycotoxins are a modifiable cause of dementia and the metabolic syndrome in mice and humans.

Age-associated dementia and Alzheimer's disease (AD) are currently epidemic. Neither their cause nor connection to the metabolic syndrome (MS) is clear. Suppression of deacetylase survival factor sirtuin 1 (SIRT1), a key host defense, is a central feature of AD. Age-related MS and diabetes are also causally associated with suppressed SIRT1 partly due to oxidant glycotoxins [advanced glycation end products (AGEs)]. Changes in the modern diet include excessive nutrient-bound AGEs, such as neurotoxic methyl-glyoxal derivatives (MG). To determine whether dietary AGEs promote AD, we evaluated WT mice pair-fed three diets throughout life: low-AGE (MG(-)), MG-supplemented low-AGE (MG(+)), and regular (Reg) chow. Older MG(+)-fed mice, similar to old Reg controls, developed MS, increased brain amyloid-ß42, deposits of AGEs, gliosis, and cognitive deficits, accompanied by suppressed SIRT1, nicotinamide phosphoribosyltransferase, AGE receptor 1, and PPARγ. These changes were not due to aging or caloric intake, as neither these changes nor the MS were present in age-matched, pair-fed MG(-) mice. The mouse data were enhanced by significant temporal correlations between high circulating AGEs and impaired cognition, as well as insulin sensitivity in older humans, in whom dietary and serum MG levels strongly and inversely associated with SIRT1 gene expression. The data identify a specific AGE (MG) as a modifiable risk factor for AD and MS, possibly acting via suppressed SIRT1 and other host defenses, to promote chronic oxidant stress and inflammation. Because SIRT1 deficiency in humans is both preventable and reversible by AGE reduction, a therapeutic strategy that includes AGE reduction may offer a new strategy to combat the epidemics of AD and MS.

Mitochondrial dysfunction and ß-cell failure in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is the most common human endocrine disease and is characterized by peripheral insulin resistance and pancreatic islet ß-cell failure. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor to ß-cell failure in the evolution of T2DM. As reviewed elsewhere, reactive oxygen species (ROS) produced by ß-cell mitochondria as a result of metabolic stress activate several stress-response pathways. This paper focuses on mechanisms whereby ROS affect mitochondrial structure and function and lead to ß-cell failure. ROS activate UCP2, which results in proton leak across the mitochondrial inner membrane, and this leads to reduced ß-cell ATP synthesis and content, which is a critical parameter in regulating glucose-stimulated insulin secretion. In addition, ROS oxidize polyunsaturated fatty acids in mitochondrial cardiolipin and other phospholipids, and this impairs membrane integrity and leads to cytochrome c release into cytosol and apoptosis. Group VIA phospholipase A2 (iPLA2ß) appears to be a component of a mechanism for repairing mitochondrial phospholipids that contain oxidized fatty acid substituents, and genetic or acquired iPLA2ß-deficiency increases ß-cell mitochondrial susceptibility to injury from ROS and predisposes to developing T2DM. Interventions that attenuate ROS effects on ß-cell mitochondrial phospholipids might prevent or retard development of T2DM.

FTY720 normalizes hyperglycemia by stimulating ß-cell in vivo regeneration in db/db mice through regulation of cyclin D3 and p57(KIP2).

Loss of insulin-producing ß-cell mass is a hallmark of type 2 diabetes in humans and diabetic db/db mice. Pancreatic ß-cells can modulate their mass in response to a variety of physiological and pathophysiological cues. There are currently few effective therapeutic approaches targeting ß-cell regeneration although some anti-diabetic drugs may positively affect ß-cell mass. Here we show that oral administration of FTY720, a sphingosine 1-phosphate (S1P) receptor modulator, to db/db mice normalizes fasting blood glucose by increasing ß-cell mass and blood insulin levels without affecting insulin sensitivity. Fasting blood glucose remained normal in the mice even after the drug was withdrawn after 23 weeks of treatment. The islet area in the pancreases of the FTY720-treated db/db mice was more than 2-fold larger than that of the untreated mice after 6 weeks of treatment. Furthermore, BrdU incorporation assays and Ki67 staining demonstrated cell proliferation in the islets and pancreatic duct areas. Finally, islets from the treated mice exhibited a significant decrease in the level of cyclin-dependent kinase inhibitor p57(KIP2) and an increase in the level of cyclin D3 as compared with those of untreated mice, which could be reversed by the inhibition of phosphatidylinositol 3-kinase (PI3K). Our findings reveal a novel network that controls ß-cell regeneration in the obesity-diabetes setting by regulating cyclin D3 and p57(KIP2) expression through the S1P signaling pathway. Therapeutic strategies targeting this network may promote in vivo regeneration of ß-cells in patients and prevent and/or cure type 2 diabetes.

Genetic ablation of PLA2G6 in mice leads to cerebellar atrophy characterized by Purkinje cell loss and glial cell activation.

Infantile neuroaxonal dystrophy (INAD) is a progressive, autosomal recessive neurodegenerative disease characterized by axonal dystrophy, abnormal iron deposition and cerebellar atrophy. This disease was recently mapped to PLA2G6, which encodes group VI Ca(2+)-independent phospholipase A(2) (iPLA(2) or iPLA(2)ß). Here we show that genetic ablation of PLA2G6 in mice (iPLA(2)ß(-/-)) leads to the development of cerebellar atrophy by the age of 13 months. Atrophied cerebella exhibited significant loss of Purkinje cells, as well as reactive astrogliosis, the activation of microglial cells, and the pronounced up-regulation of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Moreover, glial cell activation and the elevation in TNF-α and IL-1ß expression occurred before apparent cerebellar atrophy. Our findings indicate that the absence of PLA2G6 causes neuroinflammation and Purkinje cell loss and ultimately leads to cerebellar atrophy. Our study suggests that iPLA(2)ß(-/-) mice are a valuable model for cerebellar atrophy in INAD and that early anti-inflammatory therapy may help slow the progression of cerebellar atrophy in this deadly neurodegenerative disease.

Protection of pancreatic beta-cells by group VIA phospholipase A(2)-mediated repair of mitochondrial membrane peroxidation.

Mitochondrial production of reactive oxygen species and oxidation of cardiolipin are key events in initiating apoptosis. We reported that group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) localizes in and protects beta-cell mitochondria from oxidative damage during staurosporine-induced apoptosis. Here, we used iPLA(2)beta-null (iPLA(2)beta(-/-)) mice to investigate the role of iPLA(2)beta in the repair of mitochondrial membranes. We show that islets isolated from iPLA(2)beta(-/-) mice are more sensitive to staurosporine-induced apoptosis than those from wild-type littermates and that 2 wk of daily ip administration of staurosporine to iPLA(2)beta(-/-) mice impairs both the animals' glucose tolerance and glucose-stimulated insulin secretion by their pancreatic islets. Moreover, the iPLA(2)beta inhibitor bromoenol lactone caused mitochondrial membrane peroxidation and cytochrome c release, and these effects were reversed by N-acetyl cysteine. The mitochondrial antioxidant N-t-butyl hydroxylamine blocked staurosporine-induced cytochrome c release and caspase-3 activation in iPLA(2)beta(-/-) islets. Furthermore, the collapse of mitochondrial membrane potential in INS-1 insulinoma cells caused by high glucose and fatty acid levels was attenuated by overexpressing iPLA(2)beta. Interestingly, iPLA(2)beta was expressed only at low levels in islet beta-cells from obesity- and diabetes-prone db/db mice. These findings support the hypothesis that iPLA(2)beta is important in repairing oxidized mitochondrial membrane components (e.g. cardiolipin) and that this prevents cytochrome c release in response to stimuli that otherwise induce apoptosis. The low iPLA(2)beta expression level in db/db mouse beta-cells may render them vulnerable to injury by reactive oxygen species.

Advanced glycation end products inhibit glucose-stimulated insulin secretion through nitric oxide-dependent inhibition of cytochrome c oxidase and adenosine triphosphate synthesis.

Advanced glycation end products (AGEs) are implicated in diabetic complications. However, their role in beta-cell dysfunction is less clear. In this study we examined the effects of AGEs on islet function in mice and in isolated islets. AGE-BSA or BSA was administered ip to normal mice twice a day for 2 wk. We showed that AGE-BSA-treated mice exhibited significantly higher glucose levels and lower insulin levels in response to glucose challenge than did BSA-treated mice, although there were no significant differences in insulin sensitivity and islet morphology between two groups. Glucose-stimulated insulin secretion by islets of the AGE-BSA-treated mice or AGE-BSA-treated normal islets was significantly lower than that by islets isolated from the BSA-treated mice or BSA-treated normal islets. Furthermore, AGE treatment of islet beta-cells inhibited ATP production, and glimepiride, a sulfonylurea derivative, restored glucose-stimulated insulin secretion. Further investigation indicated that AGEs inhibited cytochrome c oxidase activity by inducing the expression of inducible nitric oxide synthase (iNOS). Blocking the formation of nitric oxide with an iNOS selective inhibitor aminoguanidine reversed the inhibitory effects of AGEs on ATP production and insulin secretion. We conclude that AGEs inhibit cytochrome c oxidase and ATP production, leading to the impairment of glucose-stimulated insulin secretion through iNOS-dependent nitric oxide production.