Reversible high affinity inhibition of phosphofructokinase-1 by acyl-CoA: a mechanism integrating glycolytic flux with lipid metabolism.

The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki∼1 µM). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-(14)C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.

Design and characterization of tissue-mimicking gel phantoms for diffusion kurtosis imaging.

PURPOSE: The aim of this work was to create tissue-mimicking gel phantoms appropriate for diffusion kurtosis imaging (DKI) for quality assurance, protocol optimization, and sequence development. METHODS: A range of agar, agarose, and polyvinyl alcohol phantoms with concentrations ranging from 1.0% to 3.5%, 0.5% to 3.0%, and 10% to 20%, respectively, and up to 3 g of glass microspheres per 100 ml were created. Diffusion coefficients, excess kurtosis values, and relaxation rates were experimentally determined. RESULTS: The kurtosis values for the plain gels ranged from 0.05 with 95% confidence interval (CI) of (0.029,0.071) to 0.216(0.185,0.246), well below the kurtosis values reported in the literature for various tissues. The addition of glass microspheres increased the kurtosis of the gels with values up to 0.523(0.465,0.581) observed for gels with the highest concentration of microspheres. Repeat scans of some of the gels after more than 6 months of storage at room temperature indicate changes in the diffusion parameters of less than 10%. The addition of the glass microspheres reduces the apparent diffusion coefficients (ADCs) and increases the longitudinal and transverse relaxation rates, but the values remain comparable to those for plain gels and tissue, with ADCs observed ranging from 818(585,1053) × 10-6  mm2 /s to 2257(2118,2296) × 10-6  mm2 /s, R1 values ranging from 0.34(0.32,0.35) 1/s to 0.51(0.50,0.52) 1/s, and R2 values ranging from 9.69(9.34,10.04) 1/s to 33.07(27.10, 39.04) 1/s. CONCLUSIONS: Glass microspheres can be used to effectively modify diffusion properties of gel phantoms and achieve a range of kurtosis values comparable to those reported for a variety of tissues.

Submicromolar concentrations of palmitoyl-CoA specifically thioesterify cysteine 244 in glyceraldehyde-3-phosphate dehydrogenase inhibiting enzyme activity: a novel mechanism potentially underlying fatty acid induced insulin resistance.

The accumulation of fatty acids and their metabolites results in insulin resistance and reduced glucose utilization through a variety of complex mechanisms that remain incompletely understood. Herein, we demonstrate that submicromolar concentrations of palmitoyl-CoA inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) enzyme activity through the covalent thioesterification of palmitate to GAPDH. First, incubation of GAPDH with palmitoyl-CoA (0.5-5 microM) resulted in the dramatic concentration-dependent inhibition of GAPDH enzyme activity. Second, incubation of GAPDH with [(14)C]palmitoyl-CoA followed by SDS-PAGE and autoradiography identified a covalently radiolabeled adduct present at approximately 35 kDa with a stoichiometry of one molecule of palmitoyl-CoA per GAPDH tetramer. Third, mass spectrometric analyses of intact GAPDH treated with palmitoyl-CoA demonstrated the covalent addition of palmitate to the GAPDH protein. Fourth, trypsinolysis of the modified protein revealed that the peptide (232)VPTPNVSVVDLTRC*R(245) was covalently modified. Fifth, the site of palmitoylation was demonstrated to be Cys-244 by analyses of product ion mass spectra. These assignments were further substantiated using different molecular species of acyl-CoAs resulting in the anticipated changes in both the masses of adduct ions and their fragmentation patterns. Sixth, GAPDH palmitoylation was demonstrated to facilitate the translocation of GAPDH to either lipid vesicles or naturally occurring biologic membranes. Since the hallmark of lipotoxicity is the accumulation of fatty acids and their acyl-CoA metabolites in excess of a cell's ability to appropriately metabolize them, these results identify a novel mechanism potentially contributing to the insulin resistance, reduced glucose utilization, and maladaptive metabolic alterations underlying the lipotoxic state.

Comparison of canine mandibular bone regeneration by distraction osteogenesis versus acute resection and rigid external fixation.

The present study was performed (1) to explore the mechanism of skeletal healing following distraction osteogenesis of the mandible and to evaluate whether the same process is involved following acute mandibular resection and rigid external fixation, and (2) to examine the role of the periosteum in skeletal healing in both models. The study was performed using 16 mongrel dogs divided into two equal groups. In the first group, distraction of 20 mm was performed at a rate of 1 mm/day. In the second group, bone resection of 20 mm was performed, followed by rigid external fixation. The buccal periosteum was stripped in four dogs from each group, and the periosteum was left intact in the remaining four dogs. Dogs were euthanized after a survival period of either 2 or 3 months, and the new bone regenerate was evaluated. Analysis consisted of three-dimensional computed tomography scanning, histometric analysis, and immunostaining. Analysis of bone mineral content in the residual gap was conducted. Bone mineral content was increased in 3- versus 2-month survival for all groups (p < 0.05). The distracted groups had greater bone mineral content than their acutely resected counterparts, with the difference achieving statistical significance by 3-month survival (p < 0.05). Although periosteal preservation resulted in increased bone mineral content over time for all groups (p = 0.044), periosteal preservation had no significant effect on bone mineral content in the distracted groups. After periosteal stripping, however, bone mineral content was significantly increased in dogs that underwent distraction rather than acute resection and rigid external fixation (p = 0.022). Regarding histometric analysis, analysis of fibrous tissue content in the bone regenerate demonstrated that by 3 months the distracted groups had significantly less fibrous tissue in the new bone regenerate than did the acutely resected groups (p < 0.001). Regarding immunostaining, diffuse localization of transforming growth factor-beta1 was observed in all groups at 2 months, returning to nearly baseline levels by 3 months. These data demonstrate that significant bone formation in a segmental gap can be achieved after acute mandibular resection and rigid external fixation if the periosteum is preserved. However, after periosteal injury or stripping, significant bone formation can only be achieved by distraction osteogenesis. In both processes, bone formation is preceded by up-regulation of transforming growth factor-beta1.