Sequencing refractory regions in bird genomes are hotspots for accelerated protein evolution.

BACKGROUND: Approximately 1000 protein encoding genes common for vertebrates are still unannotated in avian genomes. Are these genes evolutionary lost or are they not yet found for technical reasons? Using genome landscapes as a tool to visualize large-scale regional effects of genome evolution, we reexamined this question. RESULTS: On basis of gene annotation in non-avian vertebrate genomes, we established a list of 15,135 common vertebrate genes. Of these, 1026 were not found in any of eight examined bird genomes. Visualizing regional genome effects by our sliding window approach showed that the majority of these "missing" genes can be clustered to 14 regions of the human reference genome. In these clusters, an additional 1517 genes (often gene fragments) were underrepresented in bird genomes. The clusters of "missing" genes coincided with regions of very high GC content, particularly in avian genomes, making them "hidden" because of incomplete sequencing. Moreover, proteins encoded by genes in these sequencing refractory regions showed signs of accelerated protein evolution. As a proof of principle for this idea we experimentally characterized the mRNA and protein products of four "hidden" bird genes that are crucial for energy homeostasis in skeletal muscle: ALDOA, ENO3, PYGM and SLC2A4. CONCLUSIONS: A least part of the "missing" genes in bird genomes can be attributed to an artifact caused by the difficulty to sequence regions with extreme GC% ("hidden" genes). Biologically, these "hidden" genes are of interest as they encode proteins that evolve more rapidly than the genome wide average. Finally we show that four of these "hidden" genes encode key proteins for energy metabolism in flight muscle.

GC content of vertebrate exome landscapes reveal areas of accelerated protein evolution.

BACKGROUND: Rapid accumulation of vertebrate genome sequences render comparative genomics a powerful approach to study macro-evolutionary events. The assessment of phylogenic relationships between species routinely depends on the analysis of sequence homology at the nucleotide or protein level. RESULTS: We analyzed mRNA GC content, codon usage and divergence of orthologous proteins in 55 vertebrate genomes. Data were visualized in genome-wide landscapes using a sliding window approach. Landscapes of GC content reveal both evolutionary conservation of clustered genes, and lineage-specific changes, so that it was possible to construct a phylogenetic tree that closely matched the classic "tree of life". Landscapes of GC content also strongly correlated to landscapes of amino acid usage: positive correlation with glycine, alanine, arginine and proline and negative correlation with phenylalanine, tyrosine, methionine, isoleucine, asparagine and lysine. Peaks of GC content correlated strongly with increased protein divergence. CONCLUSIONS: Landscapes of base- and amino acid composition of the coding genome opens a new approach in comparative genomics, allowing identification of discrete regions in which protein evolution accelerated over deep evolutionary time. Insight in the evolution of genome structure may spur novel studies assessing the evolutionary benefit of genes in particular genomic regions.

Endoplasmic reticulum-associated degradation of the mouse PC1/3-N222D hypomorph and human PCSK1 mutations contributes to obesity.

BACKGROUND: The proprotein convertase 1/3 (PC1/3), encoded by proprotein convertase subtilisin/kexin type 1 (PCSK1), cleaves and hence activates several orexigenic and anorexigenic proproteins. Congenital inactivation of PCSK1 leads to obesity in human but not in mice. However, a mouse model harboring the hypomorphic mutation N222D is obese. It is not clear why the mouse models differ in phenotype. METHODS: Gene expression analysis was performed with pancreatic islets from Pcsk1(N222D/N222D) mice. Subsequently, biosynthesis, maturation, degradation and activity were studied in islets, pituitary, hypothalamus and cell lines. Coimmunoprecipitation of PC1/3-N222D and human PC1/3 variants associated with obesity with the endoplasmic reticulum (ER) chaperone BiP was studied in cell lines. RESULTS: Gene expression analysis of islets of Pcsk1(N222D/N222D) mice showed enrichment of gene sets related to the proteasome and the unfolded protein response. Steady-state levels of PC1/3-N222D and in particular the carboxy-terminally processed form were strongly reduced in islets, pituitary and hypothalamus. However, impairment of substrate cleavage was tissue dependent. Proinsulin processing was drastically reduced, while processing of proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH) in pituitary was only mildly impaired. Growth hormone expression and IGF-1 levels were normal, indicating near-normal processing of hypothalamic proGHRH. PC1/3-N222D binds to BiP and is rapidly degraded by the proteasome. Analysis of human PC1/3 obesity-associated mutations showed increased binding to BiP and prolonged intracellular retention for all investigated mutations, in particular for PC1/3-T175M, PC1/3-G226R and PC1/3-G593R. CONCLUSIONS: This study demonstrates that the hypomorphic mutation in Pcsk1(N222D) mice has an effect on catalytic activity in pancreatic islets, pituitary and hypothalamus. Reduced substrate processing activity in Pcsk1(N222D/N222D) mice is due to enhanced degradation in addition to reduced catalytic activity of the mutant. PC1/3-N222D binds to BiP, suggesting impaired folding and reduced stability. Enhanced BiP binding is also observed in several human obesity-associated PC1/3 variants, suggesting a common mechanism.

Discovery of molecular pathways mediating 1,25-dihydroxyvitamin D3 protection against cytokine-induced inflammation and damage of human and male mouse islets of Langerhans.

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced ß-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to ß-cell protection against cytokine-induced ß-cell dysfunction and death. Human and mouse islets were exposed to IL-1ß and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and ß-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in ß-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic ß-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced ß-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.

Unraveling the effects of 1,25OH2D3 on global gene expression in pancreatic islets.

INTRODUCTION: Vitamin D deficiency has been linked to type 1 and 2 diabetes, whereas supplementation may prevent both diseases. However, the extent of the effects of vitamin D or its metabolites directly on pancreatic islets is still largely unknown. The aim of the present study was to investigate how active vitamin D, 1,25(OH)2D3, affects beta cells directly by establishing its effects on global gene expression in healthy murine islets. MATERIALS AND METHODS: Pancreatic islets were isolated from 2 to 3 week old C57BL/6 mice and cultured in vitro with 1,25(OH)2D3 or vehicle for 6 and 24h. Total RNA was extracted from the islets and the effects on global gene expression were analyzed using Affymetrix microarrays. RESULTS AND DISCUSSION: Exposure to 1,25(OH)2D3 compared to vehicle resulted in 306 and 151 differentially expressed genes after 6 and 24h, respectively (n=4, >1.3-fold, p<0.02). Of these 220 were up-regulated, whereas 86 displayed a decreased expression after 6h. Furthermore, expression levels were increased for 124 and decreased for 27 genes following 24h of exposure. Formation of intercellular junctions, cytoskeletal organization, and intracellular trafficking as well as lipid metabolism and ion transport were among the most affected gene classes. Effects on several genes already identified as being part of vitamin D signaling in other cell types were observed along with genes known to affect insulin release, although with our assay we were not able to detect any effects of 1,25(OH)2D3 on glucose-stimulated insulin release from healthy pancreatic islets. CONCLUSION: The effects of 1,25(OH)2D3 on the expression of cytoskeletal and intracellular trafficking genes along with genes involved in ion transport may influence insulin exocytosis. However, an effect of 1,25(OH)2D3 on insulin release could not be detected for healthy islets in contrast to islets subjected to pathological conditions such as cytokine exposure and vitamin D deficiency as suggested by other studies. Thus, in addition to previously identified tolerogenic effects on the immune system, 1,25(OH)2D3 may affect basic functions of pancreatic beta cells, with the potential to render them more resistant to the detrimental conditions encountered during type 1 and 2 diabetes. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Endometrial gene expression in the early luteal phase is impacted by mode of triggering final oocyte maturation in recFSH stimulated and GnRH antagonist co-treated IVF cycles.

STUDY QUESTION: Do differences in endometrial gene expression exist after ovarian stimulation with four different regimens of triggering final oocyte maturation and luteal phase support in the same patient? SUMMARY ANSWER: Significant differences in the expression of genes involved in receptivity and early implantation were seen between the four protocols. WHAT IS KNOWN ALREADY: GnRH agonist triggering is an alternative to hCG triggering in GnRH antagonist co-treated cycles, resulting in an elimination of early ovarian hyperstimulation syndrome. Whereas previous studies have revealed a low ongoing clinical pregnancy rate after GnRH agonist trigger due to a high early pregnancy loss rate, despite supplementation with the standard luteal phase support, more recent studies, employing a 'modified' luteal phase support including a bolus of 1500 IU hCG on the day of oocyte aspiration, have reported ongoing pregnancy rates similar to those seen after hCG triggering. STUDY DESIGN, SIZE DURATION: A prospective randomized study was performed in four oocyte donors recruited from an oocyte donation program during the period 2010-2011. PARTICIPANTS, MATERIALS, SETTING, METHODS: Four oocyte donors in a university IVF center each prospectively underwent four consecutive stimulation protocols, with different modes of triggering final oocyte maturation and a different luteal phase support, followed by endometrial biopsy on Day 5 after oocyte retrieval. The following protocols were used: (A) 10 000 IU hCG and standard luteal phase support, (B) GnRH agonist (triptorelin 0.2 mg), followed by 1500 IU hCG 35 h after triggering final oocyte maturation, and standard luteal phase support, (C) GnRH agonist (triptorelin 0.2 mg) and standard luteal phase support and (D) GnRH agonist (triptorelin 0.2 mg) without luteal phase support. Microarray data analysis was performed with GeneSpring GX 11.5 (RMA algorithm). Pathway and network analysis was performed with the gene ontology software Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com, Redwood City, CA, USA). Samples were grouped and background intensity values were removed (cutoff at the lowest 20th percentile). A one-way ANOVA test (P< 0.05) was performed with Benjamini-Hochberg multiple testing correction. MAIN RESULTS: Significant differences were seen in endometrial gene expression, related to the type of ovulation trigger and luteal phase support. However, the endometrial gene expression after the GnRH agonist trigger and a modified luteal phase support (B) was similar to the pattern seen after the hCG trigger (A). LIMITATIONS, REASONS FOR CAUTION: The study was performed in four oocyte donors only; however, it is a strength of the study that the same donor underwent four consecutive stimulation protocols within 1 year to avoid inter-individual variations. WIDER IMPLICATIONS OF THE FINDINGS: These endometrial gene-expression findings support the clinical reports of a non-significant difference in live birth rates between the GnRH agonist trigger and the hCG trigger, when the GnRH agonist trigger is followed by a bolus of 1500 IU hCG at 35 h post trigger in addition to the standard luteal phase support. STUDY FUNDING/ COMPETING INTERESTS: This study was supported by an un-restricted research grant by MSD Belgium. TRIAL REGISTRATION NUMBER: EudraCT number 2009-009429-26, protocol number 997 (P06034).

Progesterone rise on HCG day in GnRH antagonist/rFSH stimulated cycles affects endometrial gene expression.

Premature progesterone rise during gonadotrophin-releasing hormone (GnRH) antagonist cycles for IVF is a frequent phenomenon and has been associated with lower pregnancy and implantation rates. This study evaluated endometrial gene expression on the day of oocyte retrieval according to the concentration of serum progesterone on the day of human chorionic gonadotrophin (HCG) administration in GnRH-antagonist/recombinant FSH IVF cycles with fresh embryo transfer. Endometrial biopsies (n=14) were analysed with Affymetrix HG U133 Plus 2.0 Arrays. Patients were divided into three groups according to their progesterone serum concentration on the day of HCG administration: ≤ 0.9 ng/ml (group A), 1-1.5 ng/ml (group B) and >1.5 ng/ml (group C). Gene expression analysis showed a small number of significantly differentially expressed probe sets between groups A and B (five up/23 down in B) and a large difference between groups B and C (607 up/212 down; P ≤ 0.05, fold change ≥ 1.4). Validation was performed with quantitative real-time PCR on selected genes. As far as is known, this is the first study to demonstrate a distinct difference in endometrial gene expression profile between patients with a progesterone serum concentration above and below the threshold of 1.5 ng/ml on the day of HCG administration.

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy.

AIMS/HYPOTHESIS: Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. METHODS: RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. RESULTS: mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). CONCLUSIONS/INTERPRETATION: A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified.

mRNA expression analysis of cell cycle genes in islets of pregnant mice.

AIMS/HYPOTHESIS: Pregnancy requires an increase in the functional beta cell mass to match metabolic needs for insulin. To understand this adaptation at the molecular level, we undertook a time course analysis of mRNA expression in mice. METHODS: Total RNA extracted from C57Bl6/J mouse islets every 3 days during pregnancy was hybridised on commercially available expression arrays. Gene network analysis was performed and changes in functional clusters over time visualised. The function of putative novel cell cycle genes was assessed via silencing in replicating mouse insulinoma 6 (MIN6) cells. RESULTS: Gene network analysis identified a large gene cluster associated with cell cycle control (67 genes, all upregulated by ≥ 1.5-fold, p < 0.001). The number of upregulated cell cycle genes and the mRNA expression levels of individual genes peaked at pregnancy day (P)9.5. Filtering of poorly annotated genes with enhanced expression in islets at P9.5, and in MIN6 cells and thymus resulted in further studies with G7e (also known as D17H6S56E-5) and Fignl1. Gene knock-down experiments in MIN6 cells suggested that these genes are indeed involved in adequate cell cycle accomplishment. CONCLUSIONS/INTERPRETATION: A sharp peak of cell cycle-related mRNA expression in islets occurs around P9.5, after which beta cell replication is increased. As illustrated by the identification of G7e and Fignl1 in islets of pregnant mice, further study of this distinct transcriptional peak should help to unravel the complex process of beta cell replication.

Toll-like receptor 2-deficient mice are protected from insulin resistance and beta cell dysfunction induced by a high-fat diet.

AIMS/HYPOTHESIS: Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice. METHODS: Male and female littermate Tlr2(+/+) and Tlr2(-/-) mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production. RESULTS: While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2(-/-) mice were protected from the adverse effects of HFD compared with Tlr2(+/+) littermate controls. Female Tlr2(-/-) mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2(-/-) mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2(-/-) mice did not increase IL-1beta expression in response to a NEFA mixture, whereas Tlr2(+/+) control tissues did. CONCLUSION/INTERPRETATION: These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.

Testing the hypothesis of tissue selectivity: the intersection-union test and a Bayesian approach.

MOTIVATION: Finding genes that are preferentially expressed in a particular tissue or condition is a problem that cannot be solved by standard statistical testing procedures. A relatively unknown procedure that can be used is the intersection-union test (IUT). However, two disadvantages of the IUT are that it is conservative and it conveys only the information of the least differing target tissue-other tissue pair. RESULTS: We propose a Bayesian procedure that quantifies how much evidence there is in the overall expression profile for selective over-expression. In a small simulation study, it is shown that the proposed method outperforms the IUT when it comes to finding selectively expressed genes. An application to publicly available data consisting of 22 tissues shows that the Bayesian method indeed selects genes with functions that reflect the specific tissue functions. The proposed method can also be used to find genes that are underexpressed in a particular tissue. AVAILABILITY: Both MATLAB and R code that implement the IUT and the Bayesian procedure in an efficient way, can be downloaded at http://ppw.kuleuven.be/okp/software/BayesianIUT/.

Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice.

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8(-/-)) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8(-/-) beta cells and were replaced by immature, pale insulin "progranules," which were larger than in ZnT8(+/+) islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8(-/-) mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8(-/-) genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis.

BACKGROUND AND AIMS: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor alpha (anti-TNFalpha) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. METHODS: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. RESULTS: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. CONCLUSION: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations.

AIMS/HYPOTHESIS: Survival and function of insulin-secreting pancreatic beta cells are markedly altered by changes in nutrient availability. In vitro, culture in 10 rather than 2 mmol/l glucose improves rodent beta cell survival and function, whereas glucose concentrations above 10 mmol/l are deleterious. METHODS: To identify the mechanisms of such beta cell plasticity, we tested the effects of 18 h culture at 2, 5, 10 and 30 mmol/l glucose on the transcriptome of rat islets pre-cultured for 1 week at 10 mmol/l glucose using Affymetrix Rat 230 2.0 arrays. RESULTS: Culture in either 2-5 or 30 mmol/l instead of 10 mmol/l glucose markedly impaired beta cell function, while little affecting cell survival. Of about 16,000 probe-sets reliably detected in islets, some 5,000 were significantly up- or downregulated at least 1.4-fold by glucose. Analysis of these probe-sets with GeneCluster software identified ten mRNA profiles with unidirectional up- or downregulation between 2 and 10, 2 and 30, 5 and 10, 5 and 30 or 10 and 30 mmol/l glucose. It also identified eight complex V-shaped or inverse V-shaped profiles with a nadir or peak level of expression in 5 or 10 mmol/l glucose. Analysis of genes belonging to these various clusters using Onto-express and GenMAPP software revealed several signalling and metabolic pathways that may contribute to induction of beta cell dysfunction and apoptosis after culture in low- or high- vs intermediate-glucose concentration. CONCLUSIONS/INTERPRETATION: We have identified 18 distinct mRNA profiles of glucose-induced changes in islet gene mRNA levels that should help understand the mechanisms by which glucose affects beta cell survival and function under states of chronic hypo- or hyperglycaemia.

Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets.

AIMS/HYPOTHESIS: Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein response (UPR), including the integrated stress response (ISR), the latter being also activated by other types of stresses. It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly affected by pathological disruption or excessive activation of the UPR. However, whether and how physiological nutrient stimulation affects the beta cell UPR has been little investigated. MATERIALS AND METHODS: We compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca(2+) emptying with thapsigargin on the UPR (X-box binding protein [Xbp1] mRNA splicing and XBP1/activating transcription factor [ATF] 6-target gene expression) and ISR (eukaryotic translation initiation factor 2A phosphorylation, ATF4 protein levels and target gene expression) in isolated rat islets. RESULTS: Thapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30 mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10 mmol/l; (2) moderate increase parallel to the UPR between 10 and 30 mmol/l. These glucose effects occurred within 2 h, were mimicked by other metabolic substrates, but were independent of changes in Ca(2+) influx or insulin secretion. Remarkably, attenuating the glucose stimulation of protein synthesis with a low concentration of cycloheximide prevented UPR activation but not ISR reduction by high glucose. CONCLUSIONS/INTERPRETATION: Nutrient stimulation acutely activates rat islet UPR in a manner dependent on protein synthesis, while exerting complex effects on the ISR. These effects may contribute to nutrient-induced maintenance of the beta cell phenotype.

Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets.

AIMS/HYPOTHESIS: The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion. METHODS: Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K(+) channel (K(ATP)-channel) conductance. RESULTS: We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca(2+)-induced exocytosis and K(ATP)-channel conductance in single cells, were unaffected. Insulin secretion induced by alpha-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K(ATP)-independent actions of fuel secretagogues are operational, as well as the first and K(ATP)-dependent phase. CONCLUSIONS/INTERPRETATION: Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.

Type VIII adenylyl cyclase in rat beta cells: coincidence signal detector/generator for glucose and GLP-1.

AIMS/HYPOTHESIS: The secretory function of pancreatic beta cells is synergistically stimulated by two signalling pathways which mediate the effects of nutrients and hormones such as glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) or glucagon. These hormones are known to activate adenylyl cyclase in beta cells. We examined the type of adenylyl cyclase that is associated with this synergistic interaction. METHODS: Insulin release, cAMP production, adenylyl cyclase activity, mRNA and protein expression were measured in fluorescence-activated cell sorter-purified rat beta cells and in the rat beta-cell lines RINm5F, INS-1 832/13 and INS-1 832/2. RESULTS: In primary beta cells, glucagon and GLP-1 synergistically potentiate the stimulatory effect of 20 mmol/l glucose on insulin release and cAMP production. Both effects are abrogated in the presence of the L-type Ca(2+)-channel blocker verapamil. The cAMP-producing activity of adenylyl cyclase in membranes from RINm5F cells is synergistically increased by Ca(2+)-calmodulin and recombinant GTP(gamma)S-activated G(s alpha)-protein subunits. This type of regulation is characteristic for type I and type VIII AC isoforms. Consistent with this functional data, AC mRNA analysis shows abundant expression of type VI AC, four splice variants of type VIII AC and low expression level of type I AC in beta cells. Type VIII AC expression at the protein level was observed using immunoblots of RINm5F cell extracts. CONCLUSION/INTERPRETATION: This study identifies type VIII AC in insulin-secreting cells as one of the potential molecular targets for synergism between GLP-1 receptor mediated and glucose-mediated signalling.

Physiological role of AMP-activated protein kinase (AMPK): insights from knockout mouse models.

AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To understand better the physiological role of the catalytic AMPK subunit isoforms, we generated two knockout mouse models with the alpha1 (AMPK alpha 1(-/-)) and alpha 2 (AMPK alpha 2(-/-)) catalytic subunit genes deleted. No defect in glucose homoeostasis was observed in AMPK alpha 1(-/-) mice. On the other hand, AMPK alpha 2(-/-) mice presented high plasma glucose levels and low plasma insulin concentrations in the fed period and during the glucose tolerance test. Nevertheless, in isolated AMPK alpha 2(-/-) pancreatic islets, glucose-stimulated insulin secretion was not affected. Surprisingly, AMPK alpha 2(-/-) mice were insulin-resistant and had reduced muscle glycogen synthesis as assessed in vivo by the hyperinsulinaemic euglycaemic clamp procedure. Reduction of insulin sensitivity and glycogen synthesis were not dependent on the lack of AMPK in skeletal muscle, since mice expressing a dominant inhibitory mutant of AMPK in skeletal muscle were not affected and since insulin-stimulated glucose transport in incubated muscles in vitro was normal in AMPK alpha 2(-/-) muscles. Furthermore, AMPK alpha 2(-/-) mice have a higher sympathetic tone, as shown by increased catecholamine urinary excretion. Increased adrenergic tone could explain both decreased insulin secretion and insulin resistance observed in vivo in AMPK alpha 2(-/-) mice. We suggest that the alpha2 catalytic subunit of AMPK plays a major role as a fuel sensor by modulating the activity of the autonomous nervous system in vivo.

IA-2 autoantibodies predict impending type I diabetes in siblings of patients.

AIMS/HYPOTHESIS: Multiple islet autoantibody positivity is currently believed to best predict progression to Type I (insulin-dependent) diabetes mellitus. We compared its predictive value with that of positivity for a particular type of islet autoantibody, directed against the IA-2 antigen. METHODS: Autoantibodies against islet cell cytoplasm (ICA), insulin (IAA), GAD (GADA) and IA-2 (IA-2A) were measured at initial sampling in 1724 non-diabetic siblings (median age [range]:16 [0-39] years) of Type I diabetic patients with a median follow-up of 50 months. RESULTS: On initial sampling 11% of siblings were positive for one antibody type or more and 2.1% for three of more types. During follow-up, 27 antibody-positive siblings developed diabetes. Using survival analysis, the risk for clinical onset within 5 years was 34% in subjects positive for three or more types compared with 13% in those with one type or more. Progression to diabetes amounted to 12% within 5 years among siblings positive for IAA, 20% for ICA, 19% for GADA but 59% for IA-2A (p<0.001 vs absence of the respective antibody). IA-2A were detected in 1.7% of all siblings and in 56% of the prediabetic subjects on first sampling. Initial positivity for two or three antibody markers was associated with a higher progression rate in IA-2A positive as compared to IA-2A negative siblings (p=0.001). In absence of IA-2A initial positivity for another antibody (IAA, ICA or GADA) conferred a low (<10% within 5 years) risk of diabetes compared to subjects lacking this antibody. CONCLUSIONS/INTERPRETATION: In siblings of Type I diabetic patients, IA-2A positivity is a more direct predictor of impending clinical onset than multiple antibody positivity per se. Assessment of IA-2A status allows us to select subjects with homogeneously high risk of diabetes for participation in prevention trials.