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The role of cannabinoid receptors in the development and progression of diabetic neuropathy

Zhang, Fan


Fan Zhang


Diabetic neuropathy is characterised by neurodegeneration of peripheral sensory nerves, resulting in acute pain, sensory loss, and an increased risk of limb amputation. A clearer understanding of the mechanisms underlying the development and progression of diabetic neuropathy are likely to indicate new directions for the treatment of this complication of diabetes. A neuronal target for investigation is the endocannabinoid system. The aims of this project were to investigate whether the expression of cannabinoid (CB1) receptors was decreased under hyperglycaemic conditions, which may contribute to the pathogenesis and progression of diabetic neuropathy, and whether altered CB1 expression led to a decrease in CB1 signalling.

In a rat pheochromocytoma (PC12) cell line in vitro model of diabetic neuropathy, we found a reduction in total neurite length induced by nerve growth factor (50ng/ml) at 20-50mM glucose on day 6 (P<0.01 versus 5.5mM; n=70-79 from 6 independent cultures). This effect was due to raised glucose levels, used to mimic diabetic conditions, rather than any hyperosmolality effects since mannitol (50mM) gave similar results to that of the physiological 5.5mM glucose control (P=0.79). High glucose was associated with increased oxidative stress and a dose- dependent increase in IL-6 production (P<0.05).

The expression of CB1 receptors significantly decreased in PC12 cells cultured in hyperglycaemic conditions (P<0.05; n=5 for each) which corroborated with the results from an in vivo model showing a similar decline in CB1 receptors in dorsal root ganglion neurons from diabetic rats (P<0.01; n=3 for immunohistochemistry, and n=5 for Western blot). Cell viability assays conducted in parallel on day 6 confirmed that the total cell numbers were not significantly different between the various glucose concentrations (P=0.50; n=6). These results suggest that high glucose concentrations are associated with decreased expression of CB1 receptors in nerve cells.

We assessed CBi receptor function by measuring the inhibitory effect of the CB1 receptor agonist HU210 on capsaicin-induced calcium influx in PC12 cells cultured in physiological and high glucose conditions. In fluo-4-loaded cells, capsaicin evoked an increase in fluorescence («[CaHi) in a concentration-dependent manner. Application of the CB1 agonist HU210 (1uM) significantly inhibited capsaicin-induced calcium influx (P<0.01), and this inhibitory effect of HU210 was reversed by co-application of the CB1 antagonist AM251 (1uM) but not the CB2 receptor antagonist AM630 (1uM). At concentrations of HU210 below 1uM we observed a blunted inhibition of capsaicin-evoked calcium influx in cells cultured in high glucose. However, at concentrations >1uM HU210, the function of the CB1 receptor was preserved, as evidenced by a similar degree of inhibition of capsaicin-evoked calcium influx in 5.5 and 50mM glucose conditions (61% versus 59%, respectively, n=30-50, from four independent cultures).

The synthetic CB1 agonist, HU210 (0.03-30uM), was used in order to investigate CB1 receptor- mediated ‘neurite rescue’ in PC12 cells. Addition of HU210 (0.03-3uM) had no significant effect on the total neurite length in PC12 cells cultured in 5.5mM glucose (P=0.168 versus the control/vehicle; n=148-220) while an increased total neurite length was found in cells cultured in 50mM glucose in a concentration-dependent manner. The HU210 (IuM)-mediated rescue of neurite outgrowth could be blocked by AM251 (1uM) (P<0.05) but not by AM630 (1uM), confirming the role of CB1 receptors (n=131-208). In addition, HU210 significantly reduced high glucose-induced oxidative stress in PC12 cells via a CB1-independent mechanism.

These results suggest that high glucose concentrations are associated with decreased expression, but preserved function of CB1 receptors in nerve cells. Given the neuroprotective effect of cannabinoids, CB1 receptors may be an appropriate therapeutic target in preventing the neurodegenerative process in diabetes.

Thesis Type Thesis
Deposit Date Nov 22, 2021
Publicly Available Date Nov 22, 2021
Public URL
Award Date 2007


The Role Of Cannabinoid Receptors In The Development And Progression Of Diabetic Neuropathy (9.6 Mb)

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