We shall explore genetic function by a combination of genetic and functional assays. First, via expression profiling, we intend to determine which genes for mitochondrial proteins are altered in pancreatic β-cells, liver, skeletal muscle, and adipose tissue. This will be performed in material from i) humans with or without the disease, ii) in genetically or pharmacologically manipulated animals and iii) in experimentally engineered cells. Second, a proteomics approach will be used to ascertain that the changes documented in the expression profiling have a counterpart at the protein level. Third, global changes in metabolites in these same cells and tissues as well as body fluids will be assessed using metabolomics. The efforts described will be complemented by an extensive array of functional studies of mitochondrial function such as rhodamine123 imaging if mitochondrial membrane potential in conjunction with advanced imaging techniques. Age- and disease-associated changes in mitochondrial function should also impact on the ability of glucose to close ATP-regulated K+ channels in the pancreatic β cells. There is an age-dependent shift of the IC50 value for glucose-induced inhibition of the KATP channels in mouse β-cells 38. It is essential to determine if this also occurs in human β-cells. Such a decrease may increase disease risk in individuals that are genetically predisposed to develop T2DM.

Further, we will use a mouse model with a mitchondrial DNA mutation which interacts with a polymorphism in the UCP2 gene. Both the mitochondrial polymorphism and the UCP2 mutation control metabolic and inflammatory traits and are thought to operate by enhancing the production of ROS within cells. We will crossbreed these mutants with the Ncf1 mutated mouse and study its role in both T1D and T2D. These studies will be performed in cooperation with Dr Saleh Ibrahim, Rostock, who identified both the mitochondria mutation and the UCP2 polymorphism. The recruitment of Dr Ibrahim to Lund is envisaged if this Initiative meets with success.

If mitochondrial genes are among the panel of 50 SNPs identified in the genome wide scan these will be explored in detail for their influence on mitochondrial function and whether genetic variations influence gene-expression and mitochondrial function in human target tissues.

We will elucidate the influence of epigenetic factors on the age-related decline in mitochondrial function by studying DNA methylation using both gene specific (bisulfate sequencing) and global approaches (e.g. restriction landmark genomic scanning). We will also study whether coordinated down-regulation of genes regulating oxidative phosphorylation seen in the prediabetic state is reversible by exercise.

Together, the efforts described above serve to identify candidate genes and pathways involved in the pathogenesis of T2D. In a next step, the candidates will be over-expressed or knocked out/down, using transgenic techniques in vivo or RNA interference in vitro. The aim of this extended approach is to experimentally validate the novel targets, and evaluate them for a possible role in the prevention and/or treatment of the disease.