Alternative exon splicing and differential expression in pancreatic islets reveals candidate genes and pathways implicated in early diabetes development

Type 2 diabetes (T2D) has a strong genetic component. Most of the gene variants driving the pathogenesis of T2D seem to target pancreatic β-cell function. To identify novel gene variants acting at early stage of the disease, we analyzed whole transcriptome data to identify differential expression (DE) and alternative exon splicing (AS) transcripts in pancreatic islets collected from two metabolically diverse mouse strains at 6 weeks of age after three weeks of high-fat-diet intervention. Our analysis revealed 1218 DE and 436 AS genes in islets from NZO/Hl vs C3HeB/FeJ. Whereas some of the revealed genes present well-established markers for β-cell failure, such as Cd36 or Aldh1a3, we identified numerous DE/AS genes that have not been described in context with β-cell function before. The gene Lgals2, previously associated with human T2D development, was DE as well as AS and localizes in a quantitative trait locus (QTL) for blood glucose on Chr.15 that we reported recently in our N2(NZOxC3H) population. In addition, pathway enrichment analysis of DE and AS genes showed an overlap of only half of the revealed pathways, indicating that DE and AS in large parts influence different pathways in T2D development. PPARG and adipogenesis pathways, two well-established metabolic pathways, were overrepresented for both DE and AS genes, probably as an adaptive mechanism to cope for increased cellular stress. Our results provide guidance for the identification of novel T2D candidate genes and demonstrate the presence of numerous AS transcripts possibly involved in islet function and maintenance of glucose homeostasis

Biomarker discovery and redundancy reduction towards classification using a multi-factorial MALDI-TOF MS T2DM mouse model dataset

Diabetes like many diseases and biological processes is not mono-causal. On the one hand multifactorial studies with complex experimental design are required for its comprehensive analysis. On the other hand, the data from these studies often include a substantial amount of redundancy such as proteins that are typically represented by a multitude of peptides. Coping simultaneously with both complexities (experimental and technological) makes data analysis a challenge for Bioinformatics

Physical activity attenuates postprandial hyperglycaemia in homozygous TBC1D4 loss-of-function mutation carriers

Aims/hypothesis The common muscle-specific TBC1D4 p.Arg684Ter loss-of-function variant defines a subtype of non-autoimmune diabetes in Arctic populations. Homozygous carriers are characterised by elevated postprandial glucose and insulin levels. Because 3.8% of the Greenlandic population are homozygous carriers, it is important to explore possibilities for precision medicine. We aimed to investigate whether physical activity attenuates the effect of this variant on 2 h plasma glucose levels after an oral glucose load. Methods In a Greenlandic population cohort (n = 2655), 2 h plasma glucose levels were obtained after an OGTT, physical activity was estimated as physical activity energy expenditure and TBC1D4 genotype was determined. We performed TBC1D4–physical activity interaction analysis, applying a linear mixed model to correct for genetic admixture and relatedness. Results Physical activity was inversely associated with 2 h plasma glucose levels (β[main effect of physical activity] −0.0033 [mmol/l] / [kJ kg−1 day−1], p = 6.5 × 10−5), and significantly more so among homozygous carriers of the TBC1D4 risk variant compared with heterozygous carriers and non-carriers (β[interaction] −0.015 [mmol/l] / [kJ kg−1 day−1], p = 0.0085). The estimated effect size suggests that 1 h of vigorous physical activity per day (compared with resting) reduces 2 h plasma glucose levels by an additional ~0.7 mmol/l in homozygous carriers of the risk variant. Conclusions/interpretation Physical activity improves glucose homeostasis particularly in homozygous TBC1D4 risk variant carriers via a skeletal muscle TBC1 domain family member 4-independent pathway. This provides a rationale to implement physical activity as lifestyle precision medicine in Arctic populations. Data repository The Greenlandic Cardio-Metabochip data for the Inuit Health in Transition study has been deposited at the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/dacs/EGAC00001000736) under accession EGAD00010001428

Two New Loci for Body-Weight Regulation Identified in a Joint Analysis of Genome-Wide Association Studies for Early-Onset Extreme Obesity in French and German Study Groups

Meta-analyses of population-based genome-wide association studies (GWAS) in adults have recently led to the detection of new genetic loci for obesity. Here we aimed to discover additional obesity loci in extremely obese children and adolescents. We also investigated if these results generalize by estimating the effects of these obesity loci in adults and in population-based samples including both children and adults. We jointly analysed two GWAS of 2,258 individuals and followed-up the best, according to lowest p-values, 44 single nucleotide polymorphisms (SNP) from 21 genomic regions in 3,141 individuals. After this DISCOVERY step, we explored if the findings derived from the extremely obese children and adolescents (10 SNPs from 5 genomic regions) generalized to (i) the population level and (ii) to adults by genotyping another 31,182 individuals (GENERALIZATION step). Apart from previously identified FTO, MC4R, and TMEM18, we detected two new loci for obesity: one in SDCCAG8 (serologically defined colon cancer antigen 8 gene; p = 1.85610 x 10(-8) in the DISCOVERY step) and one between TNKS (tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase gene) and MSRA (methionine sulfoxide reductase A gene; p = 4.84 x 10(-7)), the latter finding being limited to children and adolescents as demonstrated in the GENERALIZATION step. The odds ratios for early-onset obesity were estimated at similar to 1.10 per risk allele for both loci. Interestingly, the TNKS/MSRA locus has recently been found to be associated with adult waist circumference. In summary, we have completed a meta-analysis of two GWAS which both focus on extremely obese children and adolescents and replicated our findings in a large followed-up data set. We observed that genetic variants in or near FTO, MC4R, TMEM18, SDCCAG8, and TNKS/MSRA were robustly associated with early-onset obesity. We conclude that the currently known major common variants related to obesity overlap to a substantial degree between children and adults

Positional Cloning of Zinc Finger Domain Transcription Factor Zfp69, a Candidate Gene for Obesity-Associated Diabetes Contributed by Mouse Locus Nidd/SJL

Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1) contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO) mice. A critical interval of distal chromosome 4 (2.1 Mbp) conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT–PCR, and RACE–PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a) in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box) and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lepob background, the diabetogenic Zfp69SJL allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes

Leptin Reduces the Expression and Increases the Phosphorylation of the Negative Regulators of GLUT4 Traffic TBC1D1 and TBC1D4 in Muscle of ob/ob Mice

Leptin improves insulin sensitivity in skeletal muscle. Our goal was to determine whether proteins controlling GLUT4 traffic are altered by leptin deficiency and in vivo leptin administration in skeletal muscle of wild type and ob/ob mice. Leptin-deficient ob/ob mice were divided in three groups: control, leptin-treated (1 mg/kg/d) and leptin pair-fed ob/ob mice. Microarray analysis revealed that 1,546 and 1,127 genes were regulated by leptin deficiency and leptin treatment, respectively. Among these, we identified 24 genes involved in intracellular vesicle-mediated transport in ob/ob mice. TBC1 domain family, member 1 (Tbc1d1), a negative regulator of GLUT4 translocation, was up-regulated (P = 0.001) in ob/ob mice as compared to wild types. Importantly, leptin treatment reduced the transcript levels of Tbc1d1 (P<0.001) and Tbc1d4 (P = 0.004) in the leptin-treated ob/ob as compared to pair-fed ob/ob animals. In addition, phosphorylation levels of TBC1D1 and TBC1D4 were enhanced in leptin-treated ob/ob as compared to control ob/ob (P = 0.015 and P = 0.023, respectively) and pair-fed ob/ob (P = 0.036 and P = 0.034, respectively) mice. Despite similar GLUT4 protein expression in wild type and ob/ob groups a different immunolocalization of this protein was evidenced in muscle sections. Leptin treatment increased GLUT4 immunoreactivity in gastrocnemius and extensor digitorum longus sections of leptin-treated ob/ob mice. Moreover, GLUT4 protein detected in immunoprecipitates from TBC1D4 was reduced by leptin replacement compared to control ob/ob (P = 0.013) and pair-fed ob/ob (P = 0.037) mice. Our findings suggest that leptin enhances the intracellular GLUT4 transport in skeletal muscle of ob/ob animals by reducing the expression and activity of the negative regulators of GLUT4 traffic TBC1D1 and TBC1D4