Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy

Abstract

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Fig. 1.

Rotorod performance was unaltered after 4 wk of hyperglycemia, although twitch tension of the extensor digitorum longus (EDL) muscle decreased significantly. A: rotorod performance was evaluated with an incremental velocity protocol. The end velocity at which mice remained on the rotorod did not significantly change at 1, 2, or 4 wk after the onset of hyperglycemia. B: twitch tension of the EDL muscle was reduced at 4 wk after the onset of hyperglycemia. *P < 0.05. Bars represent the mean + SE obtained from 10 mice.

Fig. 2.

Miniature end-plate current (mEPC) 90–10% decay time and amplitude increased for EDL muscles removed from diabetic mice at 2 and 4 wk after the onset of hyperglycemia. A: representative mEPCs recorded at end plates of control as well as mice having 2 or 4 wk of streptozotocin (STZ)-induced hyperglycemia. These records illustrate the increased decay time and amplitude of mEPCs during diabetes. B: representative histograms of mEPC amplitude distribution showing a shift to larger amplitudes at 2- (open bars) and 4-wk (gray bars) diabetic neuromuscular junctions (NMJs) compared with controls (black bars). C: mEPC 90–10% decay time is significantly (***P < 0.01) longer than control at 2 and 4 wk after the onset of STZ-induced hyperglycemia. Each bar represents the mean + SE obtained from 4 to 5 EDL fibers for each of 4 mice (40–200 mEPCs/fiber).

Fig. 3.

End-plate current (EPC) 90–10% decay time is prolonged for EDL muscles removed from diabetic mice. A: representative EPCs recorded for control as well as mice having 2 or 4 wk of STZ-induced hyperglycemia. B: EPC 90–10% decay time is significantly (***P < 0.01) greater than control at 2 and 4 wk after the onset of STZ-induced hyperglycemia. Bars represent the mean + SE obtained from 4 to 5 EDL fibers of 4 mice (10–11 EPCs/fiber).

Fig. 4.

End-plate sensitivity to iontophoretically applied acetylcholine (ACh) increased for muscle end plates of mice at 2 wk after the onset of STZ-induced hyperglycemia. A: representative recordings of iontophoretically evoked ACh potentials recorded at end plates of the Triangularis sterni muscle of control mice and mice with 2 wk of STZ-induced hyperglycemia. For each recording, the top trace is the end-plate potential response and the bottom trace the current injected through the iontophoretic pipette containing 3 M ACh. These records illustrate the increased amplitude and prolonged decay time of the ACh-induced potential. B: average ACh sensitivity increased at 2 wk after onset of STZ-induced hyperglycemia. ACh sensitivity was calculated as the ratio of membrane potential response to nCoul of charge passed through the ACh pipette. Bars represent ACh sensitivity (mean + SE) obtained from 14 to 19 recordings from different end plates in 3 muscles from the control and the hyperglycemic groups. **P < 0.05.

Fig. 5.

Diabetes reduced acetylcholinesterase (AChE) expression and activity of the EDL muscle. A: expression of mRNA coding for AChE declines within 4 wk of STZ-induced hyperglycemia. Left: representative agarose gel showing the products for RT-PCR analysis of AChE and GAPDH. Right: histogram expressing the fold change of AChE mRNA expression relative to control and normalized to the housekeeping gene GAPDH. Relative expression was calculated by the “ΔΔC_T method” (42) from real-time PCR data. The bar presents the mean of 6 different experiments. The error bar represents standard deviation. B: histochemical detection of AChE with the Koelle reaction for the end plate region of the EDL muscle of control and 4-wk diabetic mice. Incubation time was 30 min for all muscles. The reduced staining intensity and size for EDL of diabetic animals suggest decreased AChE activity. The black arrow within the image of the 4-wk diabetic end plate indicates discontinuity of AChE. C: biochemical determination of AChE activity in EDL muscle homogenates. The y-axis is AChE activity normalized to muscle weight (nmol·mg⁻¹·s⁻¹ thiocholine produced). Bars represent the mean + SE obtained for EDL muscles from 6 control and 9 diabetic mice. **P < 0.05.

Fig. 6.

mEPC decay time for EDL muscles of 4-wk diabetic mice is less sensitive to phenserine tartrate (PT), a selective inhibitor of AChE, and more sensitive to phenethylcymserine tartrate (PEC), a selective inhibitor of butyrylcholinesterase. A: representative mEPCs recorded from EDL muscles of control and 4-wk diabetic mice before and after in vitro application of 5 μM PEC or 5 μM PT. B: histograms of mEPC decay time (mean ± SE) for EDL muscles of control and diabetic mice before and at 30 min after exposure to 5 μM PEC or 5 μM PT. PT significantly prolonged the 90–10% decay time for mEPCs of EDL muscle preparations from both control and diabetic mice. However, PT prolonged decay time by 164 and 110% for control and diabetic preparations, respectively. Additionally, prolongation of mEPC 90–10% decay time was more apparent for muscles of diabetic mice when PEC was used. Bars represent the mean + SE obtained from 4 to 5 end plates before and after PT or PEC for each of 4 muscles from different mice. ***P < 0.01 compared with control; #P < 0.05 for the difference in decay time between control + PT and diabetic + PT. ###P < 0.01 for the difference in decay time between control + PEC and diabetic + PEC.

Fig. 7.

End plates in the EDL muscle of diabetic mice do not express immature ACh receptors (AChRs). A: representative records of mEPCs recorded from EDL muscles removed from control and 4-wk diabetic mice before and after exposure to Waglerin-1 or αA OIVA. Amplitude and decay times of mEPCs were not sensitive to 1 μM of the neonatal AChR antagonist αA OIVA. In contrast, 1 μM Waglerin-1 completely inhibited mEPCs in EDL muscles of both control and diabetic mice. B: end plates of EDL muscles of control and diabetic mice were not labeled with Alexa 647 αA OIVA applied with a protocol that strongly labeled immature AChRs at end plates of the diaphragm muscle of a 4-day-old mouse. The presence of end plates was evident in all of the preparations bathed in FITC α-bungarotoxin.

Fig. 8.

EDL muscles of db/db mice have prolonged mEPCs and reduced AChE expression. A: mEPCs in the EDL of db/db mice have a significantly prolonged 90–10% decay time relative to that of control C57 mice. Each bar represents the mean + SE obtained from 4 to 5 fibers for 4 EDL muscles excised from control and db/db mice. ***P < 0.01. B: expression of mRNA coding for AChE also declines in EDL muscle of db/db mice. The histogram represents the fold change of AChE mRNA expression relative to control and normalized to the housekeeping gene GAPDH. Relative expression was calculated by the ΔΔC_T method from real-time PCR data. The bar presents the mean of 5 different experiments. The error bar represents standard deviation. *P < 0.05.