Propagation of the Allosteric Signal in Phosphofructokinase from Bacillus stearothermophilus Examined by Methyl-Transverse Relaxation-Optimized Spectroscopy Nuclear Magnetic Resonance.

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose 6-phosphate (Fru-6-P), is allosterically regulated by the K-type inhibitor phosphoenolpyruvate (PEP). The allosteric coupling between the substrate and inhibitor is quantified by a standard coupling free energy that defines an equilibrium with the Fru-6-P-bound and PEP-bound complexes on one side and the apo form and ternary complex on the other. Methyl-transverse relaxation-optimized spectroscopy (Me-TROSY) nuclear magnetic resonance was employed to gain structural information about BsPFK in all four states of ligation relevant to the allosteric coupling. BsPFK was uniformly labeled with 15N and 2H and specifically labeled with δ-[13CH3]-isoleucine utilizing an isotopically labeled α-keto acid isoleucine precursor. Me-TROSY experiments were conducted on all four ligation states, and all 30 isoleucines, which are well dispersed throughout each subunit of the enzyme, are well-resolved in chemical shift correlation maps of 13C and 1H. Assignments for 17 isoleucines were determined through three-dimensional HMQC-NOESY experiments with [U-15N,2H];Ileδ1-[13CH3]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2H,15N,13C]-BsPFK. The assignments allowed for the mapping of resonances representing isoleucine residues to a previously determined X-ray crystallography structure. This analysis, performed for all four states of ligation, has allowed specific regions of the enzyme influenced by the binding of allosteric ligands and those involved in the propagation of the allosteric effect to be identified and distinguished from one another.

Increases in bioactive lipids accompany early metabolic changes associated with ß-cell expansion in response to short-term high-fat diet.

Pancreatic ß-cell expansion is a highly regulated metabolic adaptation to increased somatic demands, including obesity and pregnancy; adult ß cells otherwise rarely proliferate. We previously showed that high-fat diet (HFD) feeding induces mouse ß-cell proliferation in less than 1 wk in the absence of insulin resistance. Here we metabolically profiled tissues from a short-term HFD ß-cell expansion mouse model to identify pathways and metabolite changes associated with ß-cell proliferation. Mice fed HFD vs. chow diet (CD) showed a 14.3% increase in body weight after 7 days; ß-cell proliferation increased 1.75-fold without insulin resistance. Plasma from 1-wk HFD-fed mice induced ß-cell proliferation ex vivo. The plasma, as well as liver, skeletal muscle, and bone, were assessed by LC and GC mass-spectrometry for global metabolite changes. Of the 1,283 metabolites detected, 159 showed significant changes [false discovery rate (FDR) < 0.1]. The majority of changes were in liver and muscle. Pathway enrichment analysis revealed key metabolic changes in steroid synthesis and lipid metabolism, including free fatty acids and other bioactive lipids. Other important enrichments included changes in the citric acid cycle and 1-carbon metabolism pathways implicated in DNA methylation. Although the minority of changes were observed in bone and plasma (<20), increased p-cresol sulfate was increased >4 fold in plasma (the largest increase in all tissues), and pantothenate (vitamin B5) decreased >2-fold. The results suggest that HFD-mediated ß-cell expansion is associated with complex, global metabolite changes. The finding could be a significant insight into Type 2 diabetes pathogenesis and potential novel drug targets.

Loss of Free Fatty Acid Receptor 2 leads to impaired islet mass and beta cell survival.

The regulation of pancreatic ß cell mass is a critical factor to help maintain normoglycemia during insulin resistance. Nutrient-sensing G protein-coupled receptors (GPCR) contribute to aspects of ß cell function, including regulation of ß cell mass. Nutrients such as free fatty acids (FFAs) contribute to precise regulation of ß cell mass by signaling through cognate GPCRs, and considerable evidence suggests that circulating FFAs promote ß cell expansion by direct and indirect mechanisms. Free Fatty Acid Receptor 2 (FFA2) is a ß cell-expressed GPCR that is activated by short chain fatty acids, particularly acetate. Recent studies of FFA2 suggest that it may act as a regulator of ß cell function. Here, we set out to explore what role FFA2 may play in regulation of ß cell mass. Interestingly, Ffar2(-/-) mice exhibit diminished ß cell mass at birth and throughout adulthood, and increased ß cell death at adolescent time points, suggesting a role for FFA2 in establishment and maintenance of ß cell mass. Additionally, activation of FFA2 with Gαq/11-biased agonists substantially increased ß cell proliferation in in vitro and ex vivo proliferation assays. Collectively, these data suggest that FFA2 may be a novel therapeutic target to stimulate ß cell growth and proliferation.

High-fat diet-induced ß-cell proliferation occurs prior to insulin resistance in C57Bl/6J male mice.

Both short- (1 wk) and long-term (2-12 mo) high-fat diet (HFD) studies reveal enhanced ß-cell mass due to increased ß-cell proliferation. ß-Cell proliferation following HFD has been postulated to occur in response to insulin resistance; however, whether HFD can induce ß-cell proliferation independent of insulin resistance has been controversial. To examine the kinetics of HFD-induced ß-cell proliferation and its correlation with insulin resistance, we placed 8-wk-old male C57Bl/6J mice on HFD for different lengths of time and assayed the following: glucose tolerance, insulin secretion in response to glucose, insulin tolerance, ß-cell mass, and ß-cell proliferation. We found that ß-cell proliferation was significantly increased after only 3 days of HFD feeding, weeks before an increase in ß-cell mass or peripheral insulin resistance was detected. These results were confirmed by hyperinsulinemic euglycemic clamps and measurements of α-hydroxybutyrate, a plasma biomarker of insulin resistance in humans. An increase in expression of key islet-proliferative genes was found in isolated islets from 1-wk HFD-fed mice compared with chow diet (CD)-fed mice. These data indicate that short-term HFD feeding enhances ß-cell proliferation before insulin resistance becomes apparent.

An assay for small scale screening of candidate ß cell proliferative factors using intact islets.

Current protocols for screening proliferative factors for ß cells ex vivo are time-consuming, require cell lines or dissociated islets, and often entail expensive specialized screening equipment. Here we present an efficient and lower cost alternative that utilizes intact mouse islets for the initial screening of proliferative compounds and allows confirmation of ß cell proliferation from the same islets. This protocol simplifies the process, saves money, and provides a way to identify ß cell proliferative factors using equipment that is ubiquitous in most laboratories.