Glucokinase activators (GKAs) promise a new pharmacotherapy for diabetics

The glucose-phosphorylating enzyme glucokinase, a promising target for developing new antidiabetic agents, was identified through the combined efforts of basic research and human biochemical genetics. Allosteric glucokinase activators (GKAs) were discovered by high-throughput screening of a large compound library and first reported in 2003. GKAs stimulate insulin release and glucose metabolism in the liver thereby lowering blood sugar, and promising trials in humans demonstrate that they are highly effective in patients with type 2 diabetes mellitus. Many companies are now attempting to develop effective and safe GKAs for treating diabetics

GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation

AbstractGCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically ‘normal’ GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations

Functional genomics of the beta-cell: short-chain 3-hydroxyacyl-coenzyme A dehydrogenase regulates insulin secretion independent of K+ currents

Recent advances in functional genomics afford the opportunity to interrogate the expression profiles of thousands of genes simultaneously and examine the function of these genes in a high-throughput manner. In this study, we describe a rational and efficient approach to identifying novel regulators of insulin secretion by the pancreatic beta-cell. Computational analysis of expression profiles of several mouse and cellular models of impaired insulin secretion identified 373 candidate genes involved in regulation of insulin secretion. Using RNA interference, we assessed the requirements of 10 of these candidates and identified four genes (40%) as being essential for normal insulin secretion. Among the genes identified was Hadhsc, which encodes short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD), an enzyme of mitochondrial beta-oxidation of fatty acids whose mutation results in congenital hyperinsulinism. RNA interference-mediated gene suppression of Hadhsc in insulinoma cells and primary rodent islets revealed enhanced basal but normal glucose-stimulated insulin secretion. This increase in basal insulin secretion was not attenuated by the opening of the KATP channel with diazoxide, suggesting that SCHAD regulates insulin secretion through a KATP channel-independent mechanism. Our results suggest a molecular explanation for the hyperinsulinemia hypoglycemic seen in patients with SCHAD deficiency

Integration of Eukaryotic Energy Metabolism: The Intramitochondrial and Cytosolic Energy States ([ATP]f/[ADP]f[Pi])

Maintaining a robust, stable source of energy for doing chemical and physical work is essential to all living organisms. In eukaryotes, metabolic energy (ATP) production and consumption occurs in two separate compartments, the mitochondrial matrix and the cytosol. As a result, understanding eukaryotic metabolism requires knowledge of energy metabolism in each compartment and how metabolism in the two compartments is coordinated. Central to energy metabolism is the adenylate energy state ([ATP]/[ADP][Pi]). ATP is synthesized by oxidative phosphorylation (mitochondrial matrix) and glycolysis (cytosol) and each compartment provides the energy to do physical work and to drive energetically unfavorable chemical syntheses. The energy state in the cytoplasmic compartment has been established by analysis of near equilibrium metabolic reactions localized in that compartment. In the present paper, analysis is presented for energy-dependent reactions localized in the mitochondrial matrix using data obtained from both isolated mitochondria and intact tissues. It is concluded that the energy state ([ATP]f/[ADP]f[Pi]) in the mitochondrial matrix, calculated from the free (unbound) concentrations, is not different from the energy state in the cytoplasm. Corollaries are: (1) ADP in both the cytosol and matrix is selectively bound and the free concentrations are much lower than the total measured concentrations; and (2) under physiological conditions, the adenylate energy states in the mitochondrial matrix and cytoplasm are not substantially different