The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was recently identified as a new intracellular binding partner for glucokinase (GK). Therefore, we studied the importance of this interaction for the activity status of GK and glucose metabolism in insulin-producing cells by overexpression of the rat liver and pancreatic islet isoforms of PFK-2/FBPase-2. PFK-2/FBPase-2 overexpression in RINm5F-GK cells significantly increased the GK activity by 78% in cells expressing the islet isoform, by 130% in cells expressing the liver isoform, and by 116% in cells expressing a cAMP-insensitive liver S32A/H258A double mutant isoform. Only in cells overexpressing the wild-type liver PFK-2/FBPase-2 isoform was the increase of GK activity abolished by forskolin, apparently due to the regulatory site for phosphorylation by a cAMP-dependent protein kinase. In cells overexpressing any isoform of the PFK-2/FBPase-2, the increase of the GK enzyme activity was antagonized by treatment with anti–FBPase-2 antibody. Increasing the glucose concentration from 2 to 10 mmol/l had a significant stimulatory effect on the GK activity in RINm5F-GK cells overexpressing any isoform of PFK-2/FBPase-2. The interaction of GK with PFK-2/FBPase-2 takes place at glucose concentrations that are physiologically relevant for the activation of GK and the regulation of glucose-induced insulin secretion. This new mechanism of posttranslational GK regulation may also represent a new site for pharmacotherapeutic intervention in type 2 diabetes treatment.

Aims/Hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intra-cellular localization. Methods Fluorescently-tagged rat and human wild-type or P446L-GKRP and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low (5.5mmol/l) or high (25mmol/l) glucose conditions. Interaction between GCK and GKRP were measured by sensitized emission-based FRET efficiency (FRETN). Results P446L-GKRP had a decreased degree of nuclear localization, ability to sequester GCK, and direct interaction with GCK as measured by FRET compared to WT-GKRP. Decreased interaction was observed between WT-GKRP and GCK at high (25mmol/l) compared to low glucose (5.5mmol/l) but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localization or GCK sequestration. Conclusions Our study suggests the common human GKRP-P446L variant results in elevated hepatic glucose uptake and disposal by increasing active, cytosolic GCK. This would increase hepatic lipid biosynthesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk.

BackgroundThe alterations of subbasal nerve plexus (SBP) innervation and corneal sensation were estimated non-invasively and compared with the values in healthy volunteers. Additionally, this study addressed the relation of SBP changes to the retinal status, glycemic control and diabetes duration.Methodology/Principal FindingsEighteen eyes of diabetic patients with peripheral diabetic neuropathy aged 68.8±8.8 years and twenty eyes of healthy volunteers aged 66.3±13.3 yrs. were investigated with in vivo confocal laser-scanning microscopy (CLSM). An adapted algorithm for image analysis was used to quantify the morphological and topological properties of SBP. These properties were correlated to incidence of diabetic retinopathy (DR) and corneal sensation (Cochet-Bonnet esthesiometer). The developed algorithm allows a fully automated analysis of pre-segmented SBP structures. Altogether, 10 parameters were analysed, and all of them revealed significant differences between diabetic patients and healthy volunteers. The nerve fibre density, total fibre length and nerve branches were found to be significantly lower in patients with diabetes than those of control subjects (nerve fibre density 0.006±0.002 vs. 0.020±0.007 mm/mm2; total fibre length 6223±2419 vs. 19961±6553 µm; nerve branches 25.3±28.6 vs. 141.9±85.7 in healthy volunteers). Also the corneal sensation was significantly lower in diabetic group when compared to controls (43±11 vs. 59±18 mm). There was found no difference in SBP morphology or corneal sensation in the subgroups with (DR) or without (NDR) diabetic retinopathy.Conclusions/SignificanceSBP parameters were significantly reduced in diabetic patients, compared to control group. Interestingly, the SBP impairment could be shown even in the diabetic patients without DR. Although automatic adapted image analysis simplifies the evaluation of in vivo CLSM data, image acquisition and quantitative analysis should be optimised for the everyday clinical practice.

Hyperglycemia leads to corneal DC infiltration, and obesity aggravates this immune response. The direct contact between DCs and the SNP can be assumed to be a trigger of nerve fiber damage and thus a contributing factor to polyneuropathy in diabetic corneas.

Mutations in mitochondrial DNA (mtDNA) cause a variety of pathologic phenotypes. In this study, we used conplastic mouse strains to characterize the impact of a mtDNA mutation in the Atp8 gene on β-cell function, reactive oxygen species (ROS) generation, β-cell mass, and glucose metabolism in response to high-fat diet (HFD). In comparison with B6-mt(AKR) controls, the B6-mt(FVB) strain carries a point mutation of the mtDNA-coded Atp8 gene (ATP synthase), leading to a fragmentated mitochondrial phenotype. Isolated pancreatic islets from 3-month-old B6-mt(FVB) mice showed increased mitochondrial generation of ROS, reduced cellular ATP levels, reduced glucose-induced insulin secretion, higher susceptibility to palmitate stress, and pathological morphology of mitochondria. ROS generation in β-cells was not affected by changes of the ambient glucose concentrations. Feeding a HFD for 3 months resulted in impaired glucose tolerance in B6-mt(FVB) mice but not in B6-mt(AKR) controls. In B6-mt(FVB) animals, glucose intolerance positively correlated with gain of body weight. Serum insulin levels and β-cell mass significantly increased in B6-mt(FVB) mice after a 3-month HFD. The data indicate that the mutation in the Atp8 gene induces mitochondrial dysfunction in β-cells with concomitant impairment of secretory responsiveness. This mitochondrial dysfunction induced a higher susceptibility to metabolic stressors, although this effect appeared not strictly linked to nutrient-induced ROS generation. The Atp8 gene mutation caused mitochondrial dysfunction, apparently stimulating an adaptive increase of β-cell mass in response to HFD, whereas mitochondrial ROS might have had an supportive role.