Sensory neurons derived from diabetic rats exhibit deficits in functional glycolysis and ATP that are ameliorated by IGF-1

Abstract

Objective The distal dying-back of the longest nerve fibres is a hallmark of diabetic neuropathy, and impaired provision of energy in the form of adenosine triphosphate (ATP) may contribute to this neurodegenerative process. We hypothesised that energy supplementation via glycolysis and/or mitochondrial oxidative phosphorylation is compromised in cultured dorsal root ganglion (DRG) sensory neurons from diabetic rodents, thus contributing to axonal degeneration. Functional analysis of glycolysis and mitochondrial respiration and real-time measurement of ATP levels in live cells were our specific means to test this hypothesis. Methods DRG neuron cultures from age-matched control or streptozotocin (STZ)-induced type 1 diabetic rats were used for in vitro studies. Three plasmids containing ATP biosensors of varying affinities were transfected into neurons to study endogenous ATP levels in real time. The Seahorse XF analyser was used for glycolysis and mitochondrial respiration measurements. Results Fluorescence resonance energy transfer (FRET) efficiency (YFP/CFP ratio) of the ATP biosensors AT1.03 (low affinity) and AT1.03 YEMK (medium affinity) were significantly higher than that measured using the ATP-insensitive construct AT1.03 R122/6K in both cell bodies and neurites of DRG neurons (p < 0.0001). The ATP level was homogenous along the axons but higher in cell bodies in cultured DRG neurons from both control and diabetic rats. Treatment with oligomycin (an ATP synthase inhibitor in mitochondria) decreased the ATP levels in cultured DRG neurons. Likewise, blockade of glycolysis using 2-deoxy- d -glucose (2-DG: a glucose analogue) reduced ATP levels (p < 0.001). Cultured DRG neurons derived from diabetic rats showed a diminishment of ATP levels (p < 0.01), glycolytic capacity, glycolytic reserve and non-glycolytic acidification. Application of insulin-like growth factor-1 (IGF-1) significantly elevated all the above parameters in DRG neurons from diabetic rats. Oligomycin pre-treatment of DRG neurons, to block oxidative phosphorylation, depleted the glycolytic reserve and lowered basal respiration in sensory neurons derived from control and diabetic rats. Depletion was much higher in sensory neurons from diabetic rats compared to control rats. In addition, an acute increase in glucose concentration, in the presence or absence of oligomycin, elevated parameters of glycolysis by 1.5- to 2-fold while having no impact on mitochondrial respiration. Conclusion We provide the first functional evidence for decreased glycolytic capacity in DRG neurons derived from type 1 diabetic rats. IGF-1 protected against the loss of ATP supplies in DRG cell bodies and axons in neurons derived from diabetic rats by augmenting various parameters of glycolysis and mitochondrial respiration.

A critical bioenergetic switch is regulated by IGF2 during murine cartilage development

“…IGF2 enhances OxPhos and reduces inflammation in macrophages; it also functions in osteoblast differentiation 74 , 75 . In contrast, IGF1 promotes glycolysis in neurons, multiple myeloma cells, and osteoblasts 24 , 76 , 77 . Thus, IGFs seem to regulate glucose metabolism in a context-dependent manner.…”

RNA Interference against ATP as a Gene Therapy Approach for Prostate Cancer

“…The procedure followed was that of upper single ATP aptamer transfection, and 24 h later the ATP probe fluorescence intensity was analyzed by flow cytometry. 24 All experiments were repeated at least 3 times.…”

“…Cell viability was examined using the CCK-8 assay according to the manufacturer's instructions as described earlier. 25 DU145 cells stably transfected with rAPM ATP or rAPM ctrl were seeded into a 6-well plate and cultured for different times (4,8,18,24,32, and 48 h), and then all wells were added with the CCK-8 reagent which produced a measurable color change that was quantified with a microplate reader at 450 nm. The viability of the control group was normalized as 100% survival, and the data represented three independent experiments done in triplicate.…”

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Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells

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