Metabolite profiling in plasma and tissues of ob/ob and db/db mice identifies novel markers of obesity and type 2 diabetes.

AIMS/HYPOTHESIS: Metabolomics approaches in humans have identified around 40 plasma metabolites associated with insulin resistance (IR) and type 2 diabetes, which often coincide with those for obesity. We aimed to separate diabetes-associated from obesity-associated metabolite alterations in plasma and study the impact of metabolically important tissues on plasma metabolite concentrations. METHODS: Two obese mouse models were studied; one exclusively with obesity (ob/ob) and another with type 2 diabetes (db/db). Both models have impaired leptin signalling as a cause for obesity, but the different genetic backgrounds determine the susceptibility to diabetes. In these mice, we profiled plasma, liver, skeletal muscle and adipose tissue via semi-quantitative GC-MS and quantitative liquid chromatography (LC)-MS/MS for a wide range of metabolites. RESULTS: Metabolite profiling identified 24 metabolites specifically associated with diabetes but not with obesity. Among these are known markers such as 1,5-anhydro-D-sorbitol, 3-hydroxybutyrate and the recently reported marker glyoxylate. New metabolites in the diabetic model were lysine, O-phosphotyrosine and branched-chain fatty acids. We also identified 33 metabolites that were similarly altered in both models, represented by branched-chain amino acids (BCAA) as well as glycine, serine, trans-4-hydroxyproline, and various lipid species and derivatives. Correlation analyses showed stronger associations for plasma amino acids with adipose tissue metabolites in db/db mice compared with ob/ob mice, suggesting a prominent contribution of adipose tissue to changes in plasma in a diabetic state. CONCLUSIONS/INTERPRETATION: By studying mice with metabolite signatures that resemble obesity and diabetes in humans, we have found new metabolite entities for validation in appropriate human cohorts and revealed their possible tissue of origin.

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33.

GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.

Phosphorylation regulates the assembly of chloroplast import machinery.

Chloroplast function depends on the translocation of cytosolically synthesized precursor proteins into the organelle. The recognition and transfer of most precursor proteins across the outer membrane depend on a membrane inserted complex. Two receptor components of this complex, Toc34 and Toc159, are GTPases, which can be phosphorylated by kinases present in the hosting membrane. However, the physiological function of phosphorylation is not yet understood in detail. It is demonstrated that both receptors are phosphorylated within their G-domains. In vitro, the phosphorylation of Toc34 disrupts both homo- and heterodimerization of the G-domains as determined using a phospho-mimicking mutant. In endogenous membranes this mutation or phosphorylation of the wild-type receptor disturbs the association of Toc34, but not of Toc159 with the translocation pore. Therefore, phosphorylation serves as an inhibitor for the association of Toc34 with other components of the complex and phosphorylation can now be discussed as a mechanism to exchange different isoforms of Toc34 within this ensemble.

The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures.

Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.