Histogram representation of the enhanced pause values of 205 BcA86F2.

<p>The distribution of log transformed enhanced pause values (log₂(Penh)) of BcA86F2 mice ranges from lower than the average phenotype value of C57BL/6J (solid line) to greater than average phenotype value of BcA86 (dashed line).</p

Airway responsiveness phenotype of BcA86 recombinant congenic strain

<p>. (A) Enhanced pause (Penh) in response to 15 mg/ml of methacholine was measured in A/J, C57BL/6J, BcA86 and BcA86 F1 mice. (n = 14 A/J, 12 C57BL/6J, 8 BcA86 and 28 BcA86F1). (B) Airway resistance of A/J, C57BL/6J and BcA86 strains in response to 25 mg/ml of methacholine. Resistance values were normalized by baseline readings and data is shown as the fold increase from baseline. Data are presented as mean ± SEM and statistical significance was calculated by one-way ANOVA and Bonferroni correction. n>5 per strain; _{*, **}, and _*** represent <i>p</i><0.05, <i>p</i><0.01, and <i>p</i><0.001, respectively.</p

Selection of candidate genes within quantitative trait locus with expression differences.

<p>(A, B, and C) Dot plots of candidate genes from microarray analysis. Expression of <i>Eapp</i> and <i>Snapc1</i> was higher in hyporesponsive C57BL/6J strain than both hyperresponsive strains, CSS12 and BcA86. Conversely, the expression of <i>Mettl21d</i> was higher in CSS12 and BcA86 compared to C57BL/6J. (D, E, and F) Quantitative RT-PCR validates the microarray expression differences of <i>Mettl21</i> and <i>Snapc1</i>, but not of <i>Eapp</i>. Data are presented as mean ± SEM and statistical significance was calculated by one-way ANOVA and Bonferroni correction. n = 3 per strain for microarray and n>5 per strain for PCR; _* and _*** represents <i>p</i><0.05 and <i>p</i><0.001 respectively.</p

Genetic background of mouse Chromosome 12 determines airway responsiveness phenotype.

<p>(A) Log₂(Penh) response curve to increasing doses of methacholine in A/J, C57BL/6J, BcA86, and CSS12 strains. The asterisks label the strains which are significantly different from C57BL/6J at each respective dose. (B) The airway resistance of A/J, C57BL/6J, BcA86 and CSS12 mice to 25 mg/ml of methacholine was measured by the gold standard invasive plethysmography and normalized to baseline readings. Data are presented as mean ± SEM and statistical significance was calculated by one-way ANOVA and Bonferroni correction. n>5 mice per strain for both experiments; _{*, **}, and _*** represent <i>p</i><0.05, <i>p</i><0.01 and <i>p</i><0.001, respectively.</p

Sequence changes and PROVEAN scores of coding non-synonymous SNPs found in quantitative trait loci.

<p>PROVEAN scores are in parentheses. SNPs are predicted to have functional effect by PROVEAN if they have a PROVEAN score of less than −2.5. SNPs. SNPs predicted to have functional effect are annotated as “deleterious” by PROVEAN and “probably damaging” by PolyPhen2 (bolded).</p

Association of Chromosome 12 markers with airway responsiveness in BcA86 mice.

<p>(A) Distribution of LOD scores of markers on Chromosome 12 used to genotype BcA86F2 mice. Dashed line at LOD  = 2.97 represents the 95% LOD threshold for significance generated from 10,000 permutations of randomized associations of the BcA86F2 genotype and phenotype data. Bayes 95% credible interval (CI) was used to determine the QTL confidence interval delimited by markers (grey dots) D12Mit285 at 54.6 Mbp (LOD score  = 4.68) and D12Mit158 at 82.6Mbp (LOD score  = 4.47). The most significant marker in the region is D12Mit52 (★) at 77.0Mbp with a LOD score of 5.11 and <i>p</i> = 3.68×10⁻³. (B) Distribution of log₂(Penh) among the three possible genotypes at D12Mit52 (AA = homozygous for A/J, BB = homozygous for C57BL/6J and AB = heterozygous). Groups with A/J genotype at D12Mit52 (AA and AB) have significantly greater mean log2(Penh) value than homozygous C57BL/6J group (BB) (AA: 1.264 ± 0.151, AB: 0.879 ± 0.114, and BB: 0.102 ± 0.157). Data are presented as mean ± SEM and _*** represents <i>p</i><0.001 from one-way ANOVA and Bonferroni correction.</p

Pathway analysis: Top disease or functions distinguishing hyperresponsive strains from hyporesponsive strain.

<p>Analysis was done using 105 genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104234#pone.0104234.s002" target="_blank">Table S2</a>) distinguishing hyperresponsive strains, BcA86 and CSS12, from hyporesponsive strain, C57BL/6J.</p

Significant markers from quantitative trait locus analysis.

<p>Significant markers have LOD scores greater than the 95% and 99% LOD thresholds generated from 10,000 permutations (LOD  = 2.97 and 3.65, respectively). Markers in bold are within the 95% confidence interval of the QTL.</p

Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

<div><p>Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied <i>ex-vivo</i> for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression.</p></div

Increased total PKCε levels in DIO islets.

<p>Proteins from ND, LDR and HDR islets were probed with an antibody against total PKCε (T-PKCε) and phospho-Ser⁷²⁹ PKCε (P-PKCε). Tubulin or total PKCε were used for normalization. (A) Representative western blot of T-PKCε and P-PKCε, (B) expression level of T-PKCε normalized by tubulin, (C) P-PKCε level normalized by tubulin and (D) by total PKCε. Means ± SEM of 8 (ND), 13 (LDR) and 10 (HDR) mice. *p<0.05, **p<0.01 vs ND, One-way ANOVA, Tukey post-hoc test.</p