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

GCK-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 ß-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.

Mice Carrying a Dominant-Negative Human PI3K Mutation Are Protected From Obesity and Hepatic Steatosis but Not Diabetes.

Phosphatidylinositol 3-kinase (PI3K) plays a central role in insulin signaling, glucose metabolism, cell growth, cell development, and apoptosis. A heterozygous missense mutation (R649W) in the p85α regulatory subunit gene of PI3K (PIK3R1) has been identified in patients with SHORT (Short stature, Hyperextensibility/Hernia, Ocular depression, Rieger anomaly, and Teething delay) syndrome, a disorder characterized by postnatal growth retardation, insulin resistance, and partial lipodystrophy. Knock-in mice with the same heterozygous mutation mirror the human phenotype. In this study, we show that Pik3r1 R649W knock-in mice fed a high-fat diet (HFD) have reduced weight gain and adipose accumulation. This is accompanied by reduced expression of several genes involved in lipid metabolism. Interestingly, despite the lower level of adiposity, the HFD knock-in mice are more hyperglycemic and more insulin-resistant than HFD-fed control mice. Likewise, when crossed with genetically obese ob/ob mice, the ob/ob mice carrying the heterozygous R649W mutation were protected from obesity and hepatic steatosis but developed a severe diabetic state. Together, our data demonstrate a central role of PI3K in development of obesity and fatty liver disease, separating these effects from the role of PI3K in insulin resistance and the resultant hyperglycemia.