Oxidative stress-dependent MMP-13 activity underlies glucose neurotoxicity
Introduction
Diabetes mellitus afflicts ~ 28 Million people in the United States and over 300 Million worldwide, a number that is expected to double in the coming decades.1., 2. Diabetes and pre-diabetes are associated with a number of complications, one of which is diabetic peripheral neuropathy. Approximately 50–60% of diabetic patients suffer from neuropathy throughout the course of the disease. The most prevalent type is distal symmetric neuropathy, which affects sensory fibers in the distal extremities, leading to symptoms such as numbness, pain and paresthesia (tingling). Symptoms often initiate in the large toe and progressively move toward more proximal regions.3 The incidence of neuropathy is accelerated under poor glycemic control,4 and often is already prevalent in pre-diabetic patients.5 While the etiology is incompletely understood, findings suggest that oxidative stress and lack of oxygenation underlies this condition. Another contributing factor is the loss of axon regenerative capacity. Regeneration is initially present in diabetic patients following an injury,6 however, progression of the disease reduces axonal regenerative capacity and shifts the balance toward degeneration without the prospect of recovery. So far, evidence suggests that the causes of diabetic neuropathy are multifactorial and hence there are no effective treatments besides pain management.4 Identifying druggable targets to treat diabetic neuropathy would greatly improve the lives of those affected.
Hyperglycemia promotes the production and accumulation of oxidative non-enzymatic end products that can cause tissue damage. These include advanced glycation end products (AGEs), their receptor (RAGE), as well as activating ligands. AGEs can stimulate the release of free radicals, which are thought to promote the development of peripheral neuropathy through glucose autoxidation, changes in the tissue concentrations of low molecular weight antioxidants like glutathione and vitamins A, C and E, or the inability to activate intracellular defense systems that reduce free radicals like catalase and superoxide dismutase (SOD).4 Besides nervous system-specific oxidation leading to neuronal damage,7 it is possible that additional cell types are involved in neuropathy formation. For instance, we previously demonstrated that the chemotherapeutic agent, paclitaxel, induces oxidative stress in zebrafish keratinocytes, which induces sensory axon degeneration through activation of MMP-13.8 Increased glucose oxidation has also been detected in human epidermal keratinocytes and rodent wounds and is thought to impair wound healing.9 Given that MMP-13 is expressed in wound keratinocytes and stimulated by the small ROS, hydrogen peroxide (H2O2),10 we hypothesized that H2O2 formation and MMP-13 activation due to increased glucose oxidation in keratinocytes might underlie diabetic neuropathy.
To test this, we have established a larval zebrafish model in which we induced hyperglycemia using glucose treatment. We find that glucose-treated zebrafish show reduced insulin receptor expression, display increased H2O2 levels in the skin and a progressive loss of sensory axons in the caudal fin. Glucose-treated fish moreover upregulate mmp13 expression in a ROS-dependent manner. Strikingly, pharmacological inhibition of ROS or MMP-13 rescued glucose-induced neurotoxicity in zebrafish and diabetic mice. Thus, our findings demonstrate the conservation of neuropathy mechanisms and we provide a new candidate for the treatment of this condition in humans.
Section snippets
Zebrafish husbandry
Zebrafish (Nacre, Tuebingen, Tg(isl2b:GFP),11 Tg(ins:Eco.NfsB-mCherry),12 and Tg(NF-κB:GFP)) were used. All fish were raised and bred according to NIH guidelines and handled in strict accordance with good animal practices as approved by the appropriate committee (MDI Biological Laboratory animal core IACUC numbers 13-20 and 17-04). Fish were kept on a 14:10 h light/dark cycle at 28.5 °C. Embryos and larvae were maintained in Ringers solution (pH 7) throughout the procedures. To minimize suffering,
Glucose treatment induces neurotoxicity
We used glucose supplementation in the media to induce hyperglycemia in larval zebrafish, based on previous reports.18 To validate this method, we incubated larval fish for 6 d (2–8 dpf) in glucose (Fig. 1a) and subsequently performed qPCR to assess the effects on glucose metabolism. This analysis showed that insulin and insulin receptor levels were reduced, as expected. Glucagon expression was increased, indicative of increased gluconeogenesis. The expression levels however varied substantially
Discussion
We developed a zebrafish model with which to study glucose-induced neurotoxicity in live animals using in vivo imaging. We show that glucose treatment induces sensory axon loss in the zebrafish caudal fin, similar to loss of axons in the distal extremities of diabetic patients. The neurotoxic effects of glucose can be explained by glucose-dependent increased ROS formation in the skin, which stimulates MMP-13 expression and renders the epidermis susceptible to mechanical stress (consistent with
Conclusions
Diabetic neuropathy is non-reversible and patients suffer from various complications. Because treatments are unavailable it is imperative to define the underlying mechanisms. This study investigated the role of oxidative stress and MMP-13 in glucose-induced somatosensory axon degeneration and found that both play a role in this process. We previously reported that MMP-13 activity also underlies paclitaxel(chemotherapy)-induced peripheral neuropathy where it is upregulated specifically in the
Acknowledgments
We thank Chi-Bin Chien and John Rawls (Duke University) for providing transgenic lines and reagents. We thank Novartis for providing intellectual input into the establishment of the diabetic peripheral neuropathy model. We also thank the zebrafish facility staff for their support.
Author contributions: S.R. designed the zebrafish studies, analyzed the data and wrote the paper. A.L.W., P.A.S., K.L.B., E.A.B., J.K.B., J.L.M., and A.D.P. performed the zebrafish experiments and edited the
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These authors contributed equally to the work.