Metformin ameliorates peripheral neuropathy in diabetic rats by downregulating autophagy via the AMPK pathway

doi:10.21203/rs.3.rs-2602056/v1

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Metformin ameliorates peripheral neuropathy in diabetic rats by downregulating autophagy via the AMPK pathway

https://doi.org/10.21203/rs.3.rs-2602056/v1

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Aims

Diabetic neuropathy (DN) represents an important complication of diabetes mellitus. Autophagy is considered to be potentially involved in the regulation of DN. Metformin is broadly utilized in the first-line treatment of diabetes. The present work aimed to assess whether and how metformin exerts protective effects in DN.

Methods

A diabetic neuropathy rat model induced by STZ was established. Metformin was administered to examine its effect on sciatic nerve pathology, and the possible mechanism was explored.

Results

Morphological damage was observed in sciatic nerve samples from diabetic animals, accompanied by decreased p-AMPK expression and increased LC-3 levels. Notably, metformin ameliorated the morphological changes in the sciatic nerve by downregulating autophagy via p-AMPK upregulation.

Conclusions

These results indicate that metformin attenuates peripheral neuropathy in diabetic rats by regulating autophagy.

Biological sciences/Biochemistry

Biological sciences/Cell biology

Biological sciences/Molecular biology

Health sciences/Diseases

Health sciences/Endocrinology

Health sciences/Neurology

Diabetes mellitus (DM) currently constitutes an important and growing health concern worldwide, which is life-threatening, causes disabilities, requires costly management, and reduces life expectancy [1]. In 2021, an estimated 537 million individuals had diabetes, a number predicted to rise to 643 and 783 million by 2030 and 2045, respectively [2, 3]. Estimates also show > 6.7 million individuals aged 20–79 died from diabetes-associated causes in 2021 [3]. Direct health costs for diabetes care approximated US$ 1 trillion in 2021 and are expected to rise year by year [3].

It is well-known that diabetic neuropathy (DN) constitutes an important complication of DM. About half of all individuals with diabetes, including pre-diabetes and type 1 and 2 diabetes, develop DN [4, 5]. DN symptoms differ depending on disease stage. Early DN cases mostly experience pain and hyperalgesia. With disease progression, numbness, muscle weakness, loss of balance, and foot ulcers gradually appear [6, 7]. Currently, DN treatments encompass intense glycemic control and pain relief drugs [8]. However, a recently published meta-analysis of DN trials suggested glycemic control confers no benefit to most DN cases [9]. In addition, a fast glucose level reduction may induce neuropathic pain, also referred to as treatment-induced neuropathy [9]. Therefore, the current DN therapies are insufficient.

Different molecular pathways are involved in diabetic neuropathy development. The pathological process of DN is multifactorial, with unknown underpinning mechanism. The potential mechanism includes the polyol pathway [10], hexosamine pathway [11], protein kinase C (PKC) pathway [12], synthesis of advanced glycation end-products (AGEs) [13], and elevation of pro-inflammatory cytokines [14]. Additionally, autophagy might be involved in the regulation of DN [15]. However, the specific relationship between autophagy and DN remains unelucidated.

Metformin is broadly applied in the first-line treatment of type 2 diabetes. Accumulating evidence reveals metformin exerts anti-inflammatory effects and improves endothelial function in high-fat diet-associated obesity or diabetes [16, 17]. Meanwhile, previous research has shown that metformin contributes to the regulation of endothelial cell function triggered by high glucose via the autophagy pathway [18, 19].

To determine the roles of metformin in DN, a rat model of diabetes was established, and whether and how metformin confers protection in DN induced by STZ was examined.

Experimental animals

Experiments involving animals followed the institutional guidelines, with approval from the Animal Ethics Committee of Fujian Medical University. Male Sprague Dawley rats (6 weeks old, 180 to 200 g), provided by Beijing SPF Biotechnology (China), underwent housing in individual cases under specific pathogen-free (SPF) conditions, with a 12-h photoperiod and freely available water in the Laboratory Animal Center of Fujian Medical University. After a 7-day adaptation, the animals were randomized to two groups. (1) The normal chow group (NC, n = 10) received a standard chow diet (24% protein, 66% carbohydrates and 10% fat) for 8 weeks, followed by oral administration of 0.9% saline for 8 weeks. (2) The diabetes mellitus (DM) group (DM, n = 30) was fed a high-fat diet (HFD, 20% protein, 20% carbohydrates and 60% fat; H10060, Beijing HFK Bioscience, China) for 8 weeks, and then subjected to intraperitoneal STZ induction (30 mg/kg) using citrate buffer (pH 4.5). Tail vein blood collection was performed 72 h following intraperitoneal treatment with STZ to measure blood glucose (BG) levels; BG > 16.7 mmol/L suggested successful modeling of diabetes. The DM group was randomly subdivided into the DM control (DMC, n = 15; HFD for 8 weeks and STZ treatment, followed by 0.9% saline administered orally for eight weeks) and metformin (DMM, n = 15; HFD for 8 weeks and STZ treatment, followed by metformin (400 mg/kg, Sigma-Aldrich) administered orally in 0.9% saline daily for 8 weeks) groups. The DMC and DMM groups received equal amounts of the vehicle 0.9% saline containing metformin or not. After the 16-week treatment period, euthanasia was performed by intraperitoneal treatment with sodium pentobarbital (40 mg/kg). BG levels were assessed every four weeks using a glucometer (Bayer, Leverkusen, Germany). Blood specimens were obtained by cardiac puncture. The experimental design and dosage regimens were based on previous reports of experimental DN [20–22]

Tail-flick Test

The rat tail was continuously irradiated with a light radiometer (Changchun New Industry Optoelectronic Technology, mdl-hd-635, China), and the time from the beginning of irradiation to the occurrence of a tail-flick reaction (tail-flick latency: second) was recorded. Each rat was tested thrice, and values were averaged. The upper limit of the incubation period was set at 30 seconds to avoid damaging the rat tail.

Motor Nerve Conduction Velocity (Mncv)

Sciatic nerves were stimulated with previously inserted electrodes using constant-current (10 to 20 mA) square-wave pulses (40 µs) for the generation of compound muscle action potential. A total of 3 latency-of-M wave pairs were obtained and averaged. The average latency difference (ALD) between first onset peak and maximum negative peak was considered the conduction time (CT) between the two sites. MNCV was derived as the distance separating the stimulating electrodes divided by ALD.

Sensor Nerve Conduction Velocity (Sncv)

Sciatic nerves were stimulated with previously inserted electrodes using constant-current (2 mA) square-wave pulses (40 µs) for evoking an H-reflex. Then, 6 latency pairs were obtained, to determine the minimal latency difference (MLD) between the two sites. SNCV was derived as the distance separating the stimulating electrodes divided by MLD. Stimulus digitization and capture utilized the RM6240 multi-channel signal collection system.

Histology Detection

Sciatic nerve segments (1 to 2 cm) underwent overnight fixation with 25 g/L glutaraldehyde at 4°C. This was followed by a 1-h post-fixation with 10 g/L osmium tetroxide at 4°C, dehydration, resin embedding and placement in araldite mixture. Blocks underwent polymerization at 60°C for 48 h. Semi-thin sections underwent staining with 5% uranyl acetate and lead citrate, followed by observation under an electron microscope (Hitachi, Japan) [23].

Western Blotting

Protein samples were obtained from sciatic nerves lysed with RIPA supplemented with protease inhibitors (Beyotime, China). SDS-PAGE was performed for protein separation, followed by electro-transfer onto polyvinylidene fluoride membranes (Millipore, USA). Upon blocking with 5% skimmed milk, the membranes underwent overnight incubation at 4°C with primary antibodies targeting phospho-AMPK (Thr-172), AMPK, LC3, β-actin (Cell Signaling Technology, USA), followed by chemiluminescent detection [24]. ImageJ v1.48 (NIH, USA) was utilized to quantitate densitometric signals.

Statistical analysis

Data are mean ± SEM. Group pairs and multiple groups were compared by the t-test and ANOVA, respectively, with GraphPad Prism 8.0 (GraphPad, USA). P < 0.05 indicated statistical significance.

Metformin ameliorates blood glucose in the rat model of diabetes

No significant differences were found in baseline blood glucose amounts in all SD rats. After eight weeks of HFD, blood glucose amounts were slightly higher in rats of the DM group compared with those of the NC group, but with no significant difference (p > 0.05). However, STZ administration combined with HFD induced a partial destruction of β-cells in the islet, which decreased insulin secretion, inducing diabetes mellitus in the rat model. Post-metformin treatment, blood glucose in the DMM group was controlled to a certain extent, with levels markedly reduced compared with the DMC group (p < 0.05), although still elevated in comparison with those of the NC group (Table 1 and Fig. 1).

Table 1

Blood glucose levels
Week	NC group (mmol/l)	DMC group (mmol/L)	DMM group (mmol/L)
0w	5.61 ± 0.86	5.59 ± 0,92	5.76 ± 0.54
4w	5.68 ± 1.14	6.98 ± 1.09	6.89 ± 1.18
8w	6.03 ± 1.09	8.14 ± 1.01	8.26 ± 1.03
12w	5.55 ± 1.32	18.61 ± 0.87**	14.15 ± 0.86#
16w	5.69 ± 0.84	17.03 ± 0.63**	9.84 ± 0.57##
Notes: Data are mean ± S.E.M. NC group, normal chow group; DMC group, diabetes mellitus group; DMM group, metformin group. **p < 0.01 vs. NC; #p < 0.05 vs. DMC;##p < 0.01 vs. DMC.

Metformin Ameliorates Peripheral Nerve Symptoms In Diabetic Rats

The tail-flick test was applied to assess the level and change of rat pain threshold and to evaluate neuropathic pain. As depicted in Fig. 2, tail-flick latency was significantly longer in the DMC group in comparison with the NC group (13.55 ± 2.21s vs 7.76 ± 1.26s, p < 0.01). However, after 10 weeks of metformin intervention, tail-flick latency was remarkably shorter in the DMM group in comparison with the DMC group (9.9 ± 0.85s vs 13.55 ± 2.21s, p < 0.05).

MNCV and SNCV were determined to examine the function of myelinated nerve fibers (Table 2). The conductance of motor and sensory nerves was significantly lower in the DMC group in comparison with the NC group. Meanwhile, MNCV and SNCV were higher in the DMM group in comparison with the DMC group (p < 0.05).

Table 2

MNCV and SNCV in various groups
	NC group	DMC group	DMM group
MNCV (m/s)	59.81 ± 2.38	44.58 ± 4.19 *	52.81 ± 3.56 #
SNCV (m/s)	49.2 ± 2.14	36.51 ± 3.01 **	42.08 ± 3.31 #
Notes: Data are mean ± S.E.M. MNCV, motor nerve conduction velocity; SNCV, sensor nerve conduction velocity; NC group, normal chow group; DMC group, diabetes mellitus group; DMM group, metformin group. p < 0.05 vs. NC; *p < 0.01 vs. NC; #p < 0.01 vs. DMM.

Metformin ameliorates the quantity and morphology of myelinated fibers of the sciatic nerve in diabetic rats

Nerve fibers were uniform and dense, with lamellae shaped into concentric circles In the NC group (A1–A3; Fig. 3). The axons were swollen and had no shrinkage, with aligned neurofilaments and microtubules. Schwann cells and axonal mitochondria were not swollen. The ridge of the mitochondrial inner membrane was clearly visible, and myelin protrusion was scarce.

Observations under the electron microscope revealed that sciatic nerves in the DMC group (B1–B3) had altered myelin configuration, myelin protrusion, lamella separation, neurofilaments, neurotubule accumulation and disarrangement, and various bubble-shaped defects in the myelinated axons. Schwann cells and axonal mitochondria were broadly swollen or lysed, and no ridge was observed for the mitochondrial inner membrane.

In the DMM group (C1–C3), myelinated nerve fibers had a relatively complete lamellar structure with reduced density and uniformity. Some bubble-shaped defects, lamella separation, neurofilaments, and neurotubule disarrangement were detected, but the ridge of the mitochondrial inner membrane regained its normal shape. In general, demyelination and axon damage persisted, although sciatic nerve pathology was considerably improved in the DMM group in comparison with the DMC group.

Metformin Inhibits Autophagy In The Sciatic Nerve Of Diabetic Rats

As depicted in Fig. 4, relative AMPK expression levels were similar in the DMM (0.058 ± 0.02), DMC (0.045 ± 0.01) and NC (0.066 ± 0.01) groups. However, phospho-AMPK (Thr-172) expression was decreased in the DMC group compared with the NC group, but restored after the metformin intervention. Meanwhile, in comparison with the NC group, LC-3 (whose expression reflects autophagy level) was markedly upregulated in the DMC group. After the metformin intervention, autophagy level was significantly decreased in comparison with the DMC group.

Diabetic peripheral neuropathy (DPN) is known as a prominent DM complication [25], whose prevalence may be decreased by continual improvements in clinical examination and diagnostic methods [26]. Hence, the present study aimed to explore the mechanism underpinning metformin-related alleviation of DPN. The current data demonstrated metformin alleviated peripheral neuropathy in diabetic rats by suppressing autophagy via the AMPK pathway, providing evidence that metformin is a basal treatment target in DPN.

Many pathways jointly contribute to the pathogenetic mechanism of DN. However, the precise regulatory factors involved remain undefined. Slowing the development of DN and preventing or even reversing its symptoms remains the top therapeutic strategies in this disorder.

Autophagy is a natural process in which damaged organelles and macromolecules are degraded. Due to its double-sided regulatory role, autophagy contributes to maintaining intracellular environmental homeostasis; a normal level of autophagy protects cells from environmental insults, but excessive and insufficient autophagy might cause disease [27]. In recent years, a series of cell culture and animal studies have confirmed the close relationship between autophagy and DN [28–30]. However, the specific functional relationship between autophagy and DN remains unclear.

Yerra [31] showed that autophagy exerts neuroprotective effects by decreasing the buildup of damaged organelles and proteins in nerve cells. Compared with normal cultured cells, Neuro2a (N2a) cells cultured with high glucose had reduced autophagosome formation, with lower Beclin1 and LC3-Ⅱ protein amounts. In other studies, Purkinje cells in the cerebellum of a 24-week-STZ treatment-induced DN rat model were degenerated, accompanied by progressive expansion of the axon ends, decreased autophagosome formation, reduced Lamp2 expression and LC3 Ⅱ/LC3 Ⅰ ratio, and increased aggregation of the p62 protein, which is specifically degraded by autophagy. Similarly, beclin-1 was remarkably downregulated in the sciatic nerve of STZ-induced DN rats [29, 32].

However, different views exist among scholars. For example, using in vivo experiments, Towns et al. [33] found that dorsal root ganglion neurons in STZ-induced DN rats had impaired mitochondrial function, increased apoptosis, enhanced autophagy, and increased number of autophagosomes co-located with the mitochondria in the neuronal cell body.

Metformin is the commonest drug for treating type 2 diabetes mellitus, which inhibits liver gluconeogenesis as a major mechanism [34]. Meanwhile, growing evidence suggests metformin contributes to the regulation of aging and cancer development [35, 36]. AMPK was initially described as a suppressor of liver acetyl-CoA carboxylase and HMG-CoA reductase, which are major factors controlling the biosynthetic pathways of fatty acid and cholesterol [37]. In addition, metformin exerts protective effects in DR (diabetic retinopathy), whose mechanisms are associated with AMPK-dependent and -independent pathways [38, 39]. In this study, we established that autophagy level in diabetic rats was enhanced after an eight-week metformin treatment. Although AMPK expression was not remarkably changed, autophagy level was decreased with increasing p-AMPK amounts, thus DN symptoms were ameliorated. Meanwhile, metformin also improved the number of myelinated fibers in the sciatic nerve and reversed the morphological changes of the sciatic nerve in diabetic rats.

In summary, this study unveils a probable mechanism by which metformin may suppress autophagy in the sciatic nerve via an AMPK-dependent pathway that phosphorylates AMPK at Thr-172 to improve DN. Therefore, metformin can be clinically used beyond its basic anti-diabetes potential. Indeed, its beneficial effects on diabetic complications are still being discovered. Consequently, metformin is to be administered throughout the treatment process of diabetes in patients without contraindications, regardless of the presence or absence of diabetic complications. In a next study, we will explore other potential mechanisms of metformin in DN control.

Acknowledgements

None

Author Contributions

FML and WL wrote the manuscript and carried out data collection and analysis. FML, WL, DYX, and XJD conducted the experiments. XLC participated in the development of research design and in manuscript writing. The authors approved the final version of the manuscript.

Data availability statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Competing Interests Statement

The authors declare no conflict of interest.

Funding

This work was funded by the Young and Middle-aged Scientific Research Project Fund in the health system of Fuzhou (Grant number 2020-S-wq3) and the Natural Science Foundation of Fujian Province, China (Grant number 2021J011308).

Ethics approval statement

All animal studies were conducted according to the institutional guidelines and approved by the Animal Ethics Committee of Fujian Medical University. And have therefore been performed in accordance with the recommendations in the ARRIVE guidelines. We recommend consulting the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.

Consent to participate

Not appliable.

Consent to publish

Not appliable.

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No competing interests reported.

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Metformin ameliorates peripheral neuropathy in diabetic rats by downregulating autophagy via the AMPK pathway

Status:

Version 1

Abstract

Aims

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Experimental animals

Tail-flick Test

Motor Nerve Conduction Velocity (Mncv)

Sensor Nerve Conduction Velocity (Sncv)

Histology Detection

Western Blotting

Statistical analysis

Results

Metformin ameliorates blood glucose in the rat model of diabetes

Metformin Ameliorates Peripheral Nerve Symptoms In Diabetic Rats

Metformin Inhibits Autophagy In The Sciatic Nerve Of Diabetic Rats

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

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