Diabetic peripheral neuropathy (DPN) is a long-term complication of diabetes with a complicated pathogenesis. AMP-activated protein kinase (AMPK) senses oxidative stress, and mitochondrial function plays a central role in the regulation of DPN. Here, we reported that DW14006 (2-[3-(7-chloro-6-[2′-hydroxy-(1,1′-biphenyl)-4-yl]-2-oxo-1,2-dihydroquinolin-3-yl)phenyl]acetic acid) as a direct AMPKα activator efficiently ameliorated DPN in both streptozotocin (STZ)-induced type 1 and BKS db/db type 2 diabetic mice. DW14006 administration highly enhanced neurite outgrowth of dorsal root ganglion neurons and improved neurological function in diabetic mice. The underlying mechanisms have been intensively investigated. DW14006 treatment improved mitochondrial bioenergetics profiles and restrained oxidative stress and inflammation in diabetic mice by targeting AMPKα, which has been verified by assay against the STZ-induced diabetic mice injected with adeno-associated virus 8–AMPKα–RNAi. To our knowledge, our work might be the first report on the amelioration of the direct AMPKα activator on DPN by counteracting multiple risk factors including mitochondrial dysfunction, oxidative stress, and inflammation, and DW14006 has been highlighted as a potential leading compound in the treatment of DPN.

Diabetes is a chronic metabolic disease, and long-term hyperglycemia injures the tissues and organs such as large vessels, microvessels, heart, brain, kidney, eyes, and feet (1). Microvascular complications including nephropathy, retinopathy, and neuropathy, which are major predictors of type 2 diabetes macrovascular complications, contribute highly to morbidity and mortality (2). Diabetic peripheral neuropathy (DPN) is a common microvascular complication of diabetes affecting up to 66% of patients with diabetes (24). Clinical characteristics of DPN include pain, paraesthesia, and sensory loss, which are the risk factors for burns, injuries, and foot ulceration (5,6). Unfortunately, there has not yet been an effective treatment for DPN due to its complex pathogenesis (7,8), and it is imperative to design efficient anti-DPN drugs based on new therapeutic strategies.

Multiple etiological factors are tightly linked to the development of DPN, and there is a growing recognition that physicians and DPN patients must aim to resist multiple risk factors to improve both daily care and clinical outcome (9,10). Pathologically, impaired mitochondrial bioenergetics (mtBE) is one of the main pathogenetic features of DPN. In diabetic rodents, bioenergetics profile and mitochondrial membrane potential (MMP) are aberrant in dorsal root ganglion (DRG) neurons (11), with twofold rise of reactive oxygen species (ROS) in axons (12,13). Inflammation is another vitally pathogenetic feature of DPN, as indicated by high levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in patients and mice with DPN (3,14). In addition, vascular dysfunction is also responsible for diabetic neuropathy, as demonstrated by restrained sciatic nerve blood flow and blood perfusion in diabetic rodents (15).

AMP-activated protein kinase (AMPK) monitors and modulates cellar energy expenditure and energy homeostasis. It regulates anabolic and catabolic pathways involving lipid metabolism and mitochondria-mediated regulation (7). Levels of phosphorylated AMPK (p-AMPK) and peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) expressions are decreased in diabetic mice (8,11). Recently, indirect AMPK activators have been reported to activate AMPK by stimulating the AMP/ADP:ATP ratio leading to the inhibition of mitochondrial respiration and further the reduction of mitochondrial ATP synthesis. For example, metformin and resveratrol exhibited abilities in attenuating DPN symptoms, although they both activated AMPK by inhibiting mtBE and showed potential side effects (7,11,16,17). Thus, given the potency of AMPK and the potential defects of indirect AMPK activators, we aimed to study direct AMPK activators against DPN.

Here, we report that small molecule DW14006 (2-[3-(7-chloro-6-[2′-hydroxy-(1,1′-biphenyl)-4-yl]-2-oxo-1,2-dihydroquinolin-3-yl)phenyl]acetic acid) (Fig. 1A) functioned as a direct AMPKα activator and ameliorated peripheral sensory neuropathy and neurovascular dysfunction in both streptozotocin (STZ)-induced type 1 and BKS db/db type 2 diabetic mice by counteracting multiple risk factors including mitochondrial dysfunction, oxidative stress, and inflammation. The related mechanisms were intensively investigated. In addition, the results in adeno-associated virus (AAV) 8–AMPKα–RNAi–injected diabetic mice further verified that DW14006 ameliorated DPN by activating AMPKα. Our work is expected to help better understand the mechanisms underlying the amelioration of direct AMPKα activation on DPN by counteracting multiple risk factors, and highlight the potential of DW14006 in the treatment of DPN.

Figure 1

DW14006 promoted neurite outgrowth in DRG neurons from diabetic mice by activating AMPKα. A: Structure of DW14006. B and D: β-tubulin III–immunostained sensory neurons from age-matched control (C57BL/6 mice), type 1 diabetic mice (STZ), DW14006-treated (15, 30 mg/kg) STZ mice (STZ+15, 30 mg/kg) (B), and age-matched control (db/m), type 2 diabetic mice (db/db), and DW14006-treated (15, 30 mg/kg) db/db mice (db/db+15, 30 mg/kg) (D) . C and E: Quantitative analyses for B and D. F: The neurite length of DRG neurons from AAV8-AMPKα-RNAi–injected STZ mice (STZ+AAV8-AMPKα-RNAi) was decreased compared with that of AAV8-NC–injected STZ mice (STZ+AAV8-NC). DW14006 (30 mg/kg) failed to enhance neurite outgrowth in DRG neurons from AAV8-AMPKα-RNAi–injected STZ mice (STZ+AAV8-AMPKα-RNAi). G: Quantitative analysis for F. All data were presented as mean ± SEM (n = 8). The data were normalized by the total neurite outgrowth of db/m, control, or STZ+AAV8-NC. ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

Figure 1

DW14006 promoted neurite outgrowth in DRG neurons from diabetic mice by activating AMPKα. A: Structure of DW14006. B and D: β-tubulin III–immunostained sensory neurons from age-matched control (C57BL/6 mice), type 1 diabetic mice (STZ), DW14006-treated (15, 30 mg/kg) STZ mice (STZ+15, 30 mg/kg) (B), and age-matched control (db/m), type 2 diabetic mice (db/db), and DW14006-treated (15, 30 mg/kg) db/db mice (db/db+15, 30 mg/kg) (D) . C and E: Quantitative analyses for B and D. F: The neurite length of DRG neurons from AAV8-AMPKα-RNAi–injected STZ mice (STZ+AAV8-AMPKα-RNAi) was decreased compared with that of AAV8-NC–injected STZ mice (STZ+AAV8-NC). DW14006 (30 mg/kg) failed to enhance neurite outgrowth in DRG neurons from AAV8-AMPKα-RNAi–injected STZ mice (STZ+AAV8-AMPKα-RNAi). G: Quantitative analysis for F. All data were presented as mean ± SEM (n = 8). The data were normalized by the total neurite outgrowth of db/m, control, or STZ+AAV8-NC. ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

Design and Synthesis of Direct AMPKα Activator DW14006

We designed direct AMPKα activators by the scaffold hopping and bioisosteric replacement principles. A-769662, the first small molecule AMPK activator disclosed by Abbott Laboratories, belongs to thienopyridone family (18), while compound 991 is a typical benzimidazole derivative developed by Merck and Metabasis (19,20). We designed and synthesized new quinolinone-based AMPKα activators retaining the key pharmacophores of A-769662 and compound 991 (Supplementary Fig. 1A). Compound synthesis is described in the Supplementary Material.

Activities of the synthetic compounds in activating recombinant AMPKα1β1γ1 and AMPKα2β1γ1 enzymes were assayed by traditional nonradioactive methods (21). Among the active compounds, DW14006 was finally selected for its highly agonistic activity against AMPKα (Supplementary Fig. 1B and C).

Adult Mouse DRG Neurons

Adult mouse DRG neurons were isolated from normal or diabetic mice as described (11). Neurons from normal mice were grown in defined Hams F12 medium with B27 additives including 10 mmol/L d-glucose and 10 nmol/L insulin, and neurons from diabetic mice were maintained in medium containing 25 mmol/L d-glucose (11).

Animals

All animals were received humane care, and animal-related protocols were approved by the Institutional Animal Care and Use Committees at Nanjing University of Chinese Medicine. Male C57BL/6 mice at 7 weeks of age were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and male BKS Cg-m+/+Leprdb/J (db/db) mice aged 15 weeks were purchased from Model Animal Research Center of Nanjing University. Considering that male mice developed a greater extent of diabetes-induced cognition deficits and peripheral neurovascular dysfunction compared with female mice (22), male mice were here used in animal-based assays.

Preparation of Diabetic Mice With DPN

Type 1 Diabetic Mice With DPN

Type 1 diabetic mice with DPN (STZ mice) were prepared by treating C57BL/6 mice with STZ (23). Briefly, 8-week-old C57BL/6 mice were treated with a single intraperitoneal (i.p.) injection of STZ (Sigma, St. Louis, MO) at 180 mg/kg body weight. Nondiabetic mice (control, age-matched C57BL/6 mice as control in STZ mice-related assay. n = 12) were injected with the same volume of vehicle buffer (Na-Citrate Buffer, pH 4.5). A type 1 diabetic model mouse was defined as a blood glucose level >16 mmol/L after 1-week injection of STZ, and STZ mice were defined at 12 weeks of age (4 weeks after STZ injection) through behavioral tests.

Type 2 Diabetic Mice With DPN

db/db mice aged 16 weeks were used as type 2 diabetic mice that developed features of human DPN (2). Age-matched heterozygotes mice (db/m) were used as control mice in db/db mice–related assay. The criteria used for db/db mice were mainly according to DPN phenotypes (abnormal neurological functions), including but not limited to the blood glucose level.

AAV8-AMPKα-RNAi Vector Preparation and Injection

AAV-induced AMPK knockdown–related assay in DRG tissue was performed to verify the amelioration of DW14006 on DPN through targeting AMPKα. AAV8-AMPKα-RNAi virus and its negative control (NC) vector (AAV8-NC) were purchased from Shanghai Genechem Co., Ltd. The titer of AAV8-AMPKα-RNAi was 9.27 × 1012 vector genomes/mL. After being injected with STZ for 4 weeks, the STZ-injected C57BL/6 mice were injected with AAV8-AMPKα-RNAi vector or AAV8-NC to tibialis anterior and gastrocnemius muscles (1.7 × 1011 vector genomes/mouse) using 5-μL Hamilton injector (24).

Animal Administration

DW14006 was dissolved in physiological saline with 3.5% DMSO and 3.5% tween 80 (vehicle buffer). Nondiabetic C57BL/6 mice, STZ mice, db/db mice or AAV8-AMPKα-RNAi–injected STZ mice (n = 12/group) were daily administrated with 15 or 30 mg/kg DW14006 by i.p. injection for 4 weeks. Control and db/m mice were administrated with the same volume of vehicle buffer as that for diabetic mice. AMPKα expression level was repressed in DRG tissue from STZ mice 2 weeks after being injected with AAV8-AMPKα-RNAi (6 weeks after i.p. STZ) (Supplementary Fig. 4G and H). Administration of DW14006 against STZ mice started after 6 weeks of STZ injection and hyperglycemic status lasted 6 weeks before administration. For db/db mice, DW14006 administration started at 16 weeks of age (2) and hyperglycemic status lasted more than 8 weeks before administration. Animals were killed after the last administration of DW14006 under sodium pentobarbital (5 mg/100 g) anesthesia. The experimental schedule was shown in Fig. 2A and B. LD50 of DW14006 (262.1682 mg/kg) against C57BL/6 mice (i.p.) was obtained by Karber’s method (25). The detail of LD50 calculation was listed in Supplementary Table 5.

Figure 2

DW14006 treatment improved neurological function by activating AMPKα. A and B: Animal experimental schedule of STZ and db/db mice. Wide blue arrows above the axis indicate DW14006 injection, and narrow blue arrows below the axis indicate behavioral tests (tactile allodynia and thermal sensitivity); blue stars indicate MNCV. CH: DW14006 (15, 30 mg/kg) administration improved neurological function by assays against tactile allodynia (C and D), radial heat plate test (E and F), and MNCV (G and H) in STZ and db/db mice (n = 8/group). IK: DW14006 (30 mg/kg) administration rendered no improvements on the neurological function of STZ+AAV8-AMPKα-RNAi mice. All data are presented as mean ± SEM (n = 8). **P < 0.01, ***P < 0.001 vs. control or db/m mice by two-way ANOVA with Tukey test. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. STZ or db/db mice by two-way ANOVA with Tukey test. T1DM, type 1 diabetes; T2DM, type 2 diabetes.

Figure 2

DW14006 treatment improved neurological function by activating AMPKα. A and B: Animal experimental schedule of STZ and db/db mice. Wide blue arrows above the axis indicate DW14006 injection, and narrow blue arrows below the axis indicate behavioral tests (tactile allodynia and thermal sensitivity); blue stars indicate MNCV. CH: DW14006 (15, 30 mg/kg) administration improved neurological function by assays against tactile allodynia (C and D), radial heat plate test (E and F), and MNCV (G and H) in STZ and db/db mice (n = 8/group). IK: DW14006 (30 mg/kg) administration rendered no improvements on the neurological function of STZ+AAV8-AMPKα-RNAi mice. All data are presented as mean ± SEM (n = 8). **P < 0.01, ***P < 0.001 vs. control or db/m mice by two-way ANOVA with Tukey test. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. STZ or db/db mice by two-way ANOVA with Tukey test. T1DM, type 1 diabetes; T2DM, type 2 diabetes.

Weight and Glucose Measurements

Weight and blood glucose levels of mice were weekly measured by published approaches (11). The glucometer used is ROCHE ACCU-CHEK Performa (CAT/TYP:04680464008, SN: 55407954627).

Tactile Allodynia and Thermal Sensitivity Tests

Tactile Allodynia Test

Mice stood on a metal grid with hind paws irritated with a series of Von Frey filaments (Ugo Basile, Comerio, Italy), and a paw withdrawal responding to each irritation was recorded (24,26).

Thermal Sensitivity Test

Briefly, a movable radiant heat source was placed directly under the mice plantar surface of the hind paw (Ugo Basile). The average withdrawal time of the left and right hind paws was used for statistical analysis (27).

Neurophysiological Measurement

The motor nerve conduction velocity (MNCV) of the sciatic nerve from the ankle to the sciatic notch was measured via bipolar electrodes with a supramaximal stimulus (3V) of 0.05 ms duration by published description (28).

Regional Blood Flow Velocity and Perfused Blood Vessel Distribution

After anesthesia with isoflurane (RWD, Shenzhen, China), the real-time regional velocity and distribution of blood flow and perfused blood vessel of the sciatic nerve and foot pads were detected by Laser Speckle Contrast Imaging/LSCI (RFLSI Pro; RWD). Blood perfusion areas of the sciatic nerve and foot pads were analyzed by Image J (29).

Immunohistochemistry and Image Analyses

DRG tissues of the mice were used for paraffin-embedded immunohistochemistry assay (IHC-P) by published approaches (11). The antibodies used for IHC-P were provided in Supplementary Table 1. DAPI was used to stain the nucleus. Morphometric analysis was performed by IHC-Toolbox in Image J (29).

Neurite Outgrowth Detection

Neurite outgrowth was detected by the approach described in the literature (11). DRG neurons from mice were incubated with primary antibody against neuron-specific β-tubulin isotype III (1:1,000; Sigma Aldrich), followed by incubation with fluorescein 488-conjugated secondary antibody (1:300; Proteintech). Leica fluorescence microscope equipped was used to image. Total length of axon outgrowth was quantified using Neuron J plug-in components in ImageJ software (30).

Mitochondrial Function Test

MMP Test

The integrity of the mitochondrial membrane was measured by staining with JC-1, an ideal fluorescent probe widely used to detect mitochondrial membrane potential (Beyotime Biotechnology, Shanghai, China) and tetramethylrhodamine (Invitrogen) by the published approach (11,31). Fluorescent data were analyzed with Microplate Reader (Molecular Devices, San Jose, CA).

Mitochondrial Respiration Assay

An XF96 Analyzer (Seahorse Biosciences) was used to measure the basal mitochondrial oxygen consumption, maximal respiration, spare respiratory capacity and ATP production of DRG neuron from mice by the published methods (11).

Western Blotting and ELISA

Western blot assay against the homogenates of DRG and spleen tissues was performed as previously described (8) and the details of antibodies were provided in Supplementary Table 2. The blots were imaged using a ChemiDoc MP (Bio-Rad). ELISA assay was performed by commercial kits (Nanjing Jiancheng Bioengineering Institute).

Biochemical Measurement

Total superoxidase dismutase (SOD) activity and glutathione (GSH) and malondialdehyde (MDA) levels in serum were determined using commercial kits by manufacturer’s protocols (Nanjing Jiancheng Bioengineering Institute).

Superoxide Assessment

Superoxide level was measured by the published approach (32). DRG neurons from mice were seeded in a 12-well plate for 24 h, MitoSOX red mitochondrial superoxide indicator (0.5 μmol/L) and Mito Tracker Green (90 nmol/L) (Invitrogen-Molecular Probes) were added into each well and incubated for 10 min. Fluorescence intensity was quantified with ImageJ software (30).

Pharmacokinetic Properties and Bioavailability Assay in Mice

Considering the poor oral pharmacokinetic properties of DW14006, we also optimized DW14006 by using prodrug strategy. The determined ethyl ester prodrug DW15139 with better pharmacokinetic properties (Supplementary Tables 3 and 4) has well provided leading structural information for DW14006-based anti-DPN drug discovery. In the assay, half male and half female C57BL/6 mice were administered with DW15139 by oral gavage or intravenous injection. Plasma and snap-frozen DRG were collected at eight time points for analysis of DW15139 and its hydrolysate DW14006 levels by liquid chromatography–tandem mass spectrometry assay.

siRNA Transfection

DRG neurons were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols, and nontargeting siRNA was used as a NC. Interference efficiency was detected by Western blot.

Statistical Analysis

Data were presented as mean ± SE/SD. Unpaired two-tailed Student t test was used for two-group comparison. Statistical comparisons were made by one-way ANOVA or two-way ANOVA with between-group differences identified using Tukey or Dunnett post hoc tests for at least three groups’ comparisons. Significance was defined as P < 0.05.

Data and Resource Availability

The data sets generated during the current study are available from the corresponding author upon reasonable request.

DW14006 Promoted Neurite Outgrowth of DRG Neuron by Activating AMPKα

As axonal loss contributes largely to DPN induction (33,34), we examined the potential of DW14006 in promoting neurite outgrowth of DRG neuron from mice.

As expected, DW14006 enhanced neurite outgrowth in cultured adult DRG neurons from normal (Supplementary Fig. 3A and B), STZ (Fig. 1B and C), and db/db (Fig. 1D and E) diabetic mice. As indicated in Supplementary Fig. 3C and D, treatment of AMPKα-specific inhibitor compound C (CC, 3 μmol/L, MedChemExpress) (11) obviously blocked the stimulation of DW14006 on neurite outgrowth in cultured DRG neurons from normal mice. Moreover, AMPKα knockdown by AAV8-AMPKα-RNAi deprived DW14006 of the capability in promoting neurite growth of DRG neurons from AAV8-AMPKα-RNAi–injected STZ (STZ+AAV8-AMPKα-RNAi) mice (Fig. 1F and G).

DW14006 Treatment Improved Neurological Functions of Diabetic Mice by Activating AMPKα

As indicated in Fig. 2, STZ and db/db mice both exhibited increases in 50% paw withdrawal threshold (Fig. 2C and D) and thermal response latencies (Fig. 2E and F), while decreases in MNCV (Fig. 2G and H), indicative of the typical pathological features of DPN. Obviously, DW14006 treatment (15, 30 mg/kg) ameliorated all above-mentioned DPN pathologies (Fig. 2C–H). Notably, AMPKα knockdown by AAV8-AMPKα-RNAi could block such capabilities of DW14006 in STZ+AAV8-AMPKα-RNAi mice, additionally, AMPKα knockdown alone failed to affect the sensory sensitivity and MNCV in STZ mice (Fig. 2I–K). Moreover, DW14006 had no effects on sensory sensitivity and MNCV in nondiabetic mice (Supplementary Fig. 9).

DW14006 Treatment Improved Vascular Function in Peripheral Nerve Tissue of Diabetic Mice by Activating AMPKα

As indicated in Fig. 3A–D, both STZ and db/db mice showed decreases, and DW14006-treated (15, 30 mg/kg) diabetic mice showed increases, in blood flow velocity and blood perfusion ratio of sciatic nerve and foot pad tissue. Notably, AMPKα knockdown by AAV8-AMPKα-RNAi blocked such capabilities of DW14006 in STZ+AAV8-AMPKα-RNAi mice (Supplementary Fig. 6).

Figure 3

DW14006 treatment improved neurovascular function. A: Representative images of regional blood flow and perfusion ratio in sciatic nerve and foot pad tissues of control, STZ mice, and DW14006-treated (15, 30 mg/kg) STZ mice (STZ + 15, 30 mg/kg). B: Quantitative analysis for A. C: Representative images of regional blood flow and perfusion ratios in sciatic nerve and foot pad tissues of db/m, db/db, and DW14006-treated (15, 30 mg/kg) db/db mice (db/db + 15, 30 mg/kg). D: Quantitative analysis for C. All data were normalized by the blood flow and perfusion areas in sciatic nerve and foot pad tissues from control and db/m mice presented as mean ± SEM (n = 8). Lowest blood flow ratio was indicated in blue, maximum blood flow was in red, and intermediate grading was in green or yellow. ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

Figure 3

DW14006 treatment improved neurovascular function. A: Representative images of regional blood flow and perfusion ratio in sciatic nerve and foot pad tissues of control, STZ mice, and DW14006-treated (15, 30 mg/kg) STZ mice (STZ + 15, 30 mg/kg). B: Quantitative analysis for A. C: Representative images of regional blood flow and perfusion ratios in sciatic nerve and foot pad tissues of db/m, db/db, and DW14006-treated (15, 30 mg/kg) db/db mice (db/db + 15, 30 mg/kg). D: Quantitative analysis for C. All data were normalized by the blood flow and perfusion areas in sciatic nerve and foot pad tissues from control and db/m mice presented as mean ± SEM (n = 8). Lowest blood flow ratio was indicated in blue, maximum blood flow was in red, and intermediate grading was in green or yellow. ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

DW14006 Ameliorated Mitochondrial Function of DRG Neuron From Diabetic Mice by Upregulating AMPKα/SIRT1/PGC-1α Pathway and Mitochondrial Complexes I and IV Expressions

DW14006 Improved mtBEs

Since oxygen consumption rate (OCR) is vital for mtBEs and its impairment is highly related to DPN development (11), OCR was measured in DRG neurons from mice by the Seahorse Biosciences XF96 analyzer.

OCR in DRG neurons from STZ (Fig. 4A) and db/db (Fig. 4B) mice gave low levels of basal respiration (Fig. 4C and D), ATP production (Fig. 4E and F), maximal respiration capacity (Fig. 4G and H), and spare respiratory capacity (Fig. 4I and J) compared with those from age-matched control mice. DW14006 (30 mg/kg) treatment ameliorated these effects (Fig. 4C–J), and AMPKα knockdown by AAV8-AMPKα-RNAi deprived DW14006 of such amelioration against DRG tissue from STZ+AAV8-AMPKα-RNAi mice (Fig. 4K–O).

Figure 4

DW14006 treatment improved mitochondrial respiration and MMP in DRG neurons from diabetic mice by activating AMPKα. A and B: OCR was measured at basal level with sequential addition of oligomycin (1 μmol/L), FCCP (1 μmol/L), and rotenone (1 μmol/L) + antimycin A (AA; 1 μmol/L) to DRG neurons from control, db/m, STZ, db/db, and DW14006-treated (15, 30 mg/kg) (STZ + 15, 30 mg/kg, db/db + 15, 30 mg/kg) mice. OCR data were normalized by total protein content of each well and presented as pmol/min/mg protein. CJ: Basal respiration (C and D), ATP production (E and F), maximal respiration (G and H), and spare respiration capacity (I and J) were measured. The results indicated that DW14006 treatment improved mitochondrial respiration in diabetic mice. KO: DW14006 failed to impact OCR in STZ+AAV8-AMPKα-RNAi mice. Values are presented as mean ± SEM of n = 8 replicate cultures. P and Q: Relative levels of inner MMP in neurons from control, db/m, STZ, and db/db mice as well as diabetic mice treated with DW14006 (15, 30 mg/kg). MMP was presented as a ratio of aggregate to monomer. The monomer indicates early apoptosis, and the aggregate indicates the normal state. Values are presented as mean ± SEM of n = 8 replicate cultures. *P < 0.05, **P < 0.01, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by Student t test (two groups) or one-way ANOVA with Dunnett post hoc test (three groups).

Figure 4

DW14006 treatment improved mitochondrial respiration and MMP in DRG neurons from diabetic mice by activating AMPKα. A and B: OCR was measured at basal level with sequential addition of oligomycin (1 μmol/L), FCCP (1 μmol/L), and rotenone (1 μmol/L) + antimycin A (AA; 1 μmol/L) to DRG neurons from control, db/m, STZ, db/db, and DW14006-treated (15, 30 mg/kg) (STZ + 15, 30 mg/kg, db/db + 15, 30 mg/kg) mice. OCR data were normalized by total protein content of each well and presented as pmol/min/mg protein. CJ: Basal respiration (C and D), ATP production (E and F), maximal respiration (G and H), and spare respiration capacity (I and J) were measured. The results indicated that DW14006 treatment improved mitochondrial respiration in diabetic mice. KO: DW14006 failed to impact OCR in STZ+AAV8-AMPKα-RNAi mice. Values are presented as mean ± SEM of n = 8 replicate cultures. P and Q: Relative levels of inner MMP in neurons from control, db/m, STZ, and db/db mice as well as diabetic mice treated with DW14006 (15, 30 mg/kg). MMP was presented as a ratio of aggregate to monomer. The monomer indicates early apoptosis, and the aggregate indicates the normal state. Values are presented as mean ± SEM of n = 8 replicate cultures. *P < 0.05, **P < 0.01, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by Student t test (two groups) or one-way ANOVA with Dunnett post hoc test (three groups).

DW14006 Improved MMP

Next, the potential of DW14006 in ameliorating MMP of DRG neurons was detected by using JC-1 and tetramethylrhodamine dyes (11). As indicated in Fig. 4P–Q and Supplementary Fig. 7, MMP levels were decreased in DRG neurons from diabetic mice and high glucose (HG)-treated (45 mmol/L) primary DRG neurons but increased by DW14006 treatment, implying that DW14006 protected mitochondria from apoptosis in DRG neurons.

DW14006 Upregulated the AMPKα/SIRT1/PGC-1α Pathway and Increased NDUFS3 and COX IV Expressions

Considering the potency of AMPK/SIRT1/PGC-1α (SIRT1, silent information regulator factor 2-related enzyme 1) signaling in sensing oxidative stress and mitochondrial function (8), we detected the potential regulation of DW14006 against this pathway. As indicated in Fig. 5A–D, compared with the results from control mice (control, db/m), the levels of p-AMPKα and PGC-1α in DRG homogenate were decreased from diabetic mice and increased from DW14006-treated (15, 30 mg/kg) diabetic mice, while SIRT1 expression level in DRG homogenate was unaltered from STZ mice but downregulated from db/db mice, which was in agreement with the published report (35). Notably, DW14006 (15, 30 mg/kg) treatment increased SIRT1 expression in DRG homogenate from either type of diabetic mice compared with that from vehicle-treated diabetic mice. In DRG homogenate from STZ+AAV8-AMPKα-RNAi mice, the protein expression levels of AMPKα, p-AMPKα, and SIRT1 were downregulated compared with the results from AAV8-NC–injected STZ mice (STZ + AAV8-NC), and DW14006 administration gave no influence on these protein expressions in STZ+AAV8-AMPKα-RNAi mice (Fig. 5E and F). In addition, mitochondrial complexes I (NDUFS3) and IV (COX IV) were commonly used to evaluate mtBE dysfunction (36), and our results indicated that DW14006 treatment stimulated expressions of NDUFS3 and COX IV, and AMPKα knockdown by AAV8-AMPKα-RNAi blocked the activity of DW14006 in stimulating these two complexes of DRG tissue from STZ+AAV8-AMPKα-RNAi mice (Supplementary Fig. 4).

Figure 5

DW14006 stimulated AMPKα/SIRT1/PGC-1α signaling by activating AMPKα in diabetic mice. A: Quantitative immunoblot analysis revealed that the expressions of p-AMPK, phosphorylated acetyl CoA carboxylase (p-ACC), SIRT1, and PGC-1α were reduced in STZ mice and upregulated in the DW14006-treated (15, 30 mg/kg) STZ mice (STZ+15, 30 mg/kg). B: Quantitative analyses for A. C: Quantitative immunoblot analysis revealed that the expressions of p-AMPK, p-ACC, SIRT1, and PGC-1α were reduced in db/db mice (db/db) and upregulated in DW14006-treated (15, 30 mg/kg) db/db mice (db/db + 15, 30 mg/kg) vs. db/m mice. D: Quantitative analyses for C. E and F: DW14006 (30 mg/kg) had no effects on AMPKα/SIRT1/PGC-1α signaling in STZ+AAV8-AMPKα-RNAi mice. F: Quantitative analyses for E. All expressions were normalized to total extracellular signal–regulated kinase (T-ERK) level. T-ERK remained unchanged. Values are presented as mean ± SD of n = 3. *P < 0.05, **P < 0.01, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

Figure 5

DW14006 stimulated AMPKα/SIRT1/PGC-1α signaling by activating AMPKα in diabetic mice. A: Quantitative immunoblot analysis revealed that the expressions of p-AMPK, phosphorylated acetyl CoA carboxylase (p-ACC), SIRT1, and PGC-1α were reduced in STZ mice and upregulated in the DW14006-treated (15, 30 mg/kg) STZ mice (STZ+15, 30 mg/kg). B: Quantitative analyses for A. C: Quantitative immunoblot analysis revealed that the expressions of p-AMPK, p-ACC, SIRT1, and PGC-1α were reduced in db/db mice (db/db) and upregulated in DW14006-treated (15, 30 mg/kg) db/db mice (db/db + 15, 30 mg/kg) vs. db/m mice. D: Quantitative analyses for C. E and F: DW14006 (30 mg/kg) had no effects on AMPKα/SIRT1/PGC-1α signaling in STZ+AAV8-AMPKα-RNAi mice. F: Quantitative analyses for E. All expressions were normalized to total extracellular signal–regulated kinase (T-ERK) level. T-ERK remained unchanged. Values are presented as mean ± SD of n = 3. *P < 0.05, **P < 0.01, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

Additionally, it was noted that DW14006 rendered no effects on the expressions of phosphorylated nuclear factor-κB (p-NF-κB) and PGC-1α after treatment with SIRT1 siRNA in HG-cultured (45 mmol/L) DRG neurons from normal mice (Fig. 6).

Figure 6

DW14006 had no effects on the expressions of p-NF-κB and PGC-1α in HG-cultured DRG neurons treated with SIRT1 siRNA. A and B: Western blot and its quantification results demonstrated that SIRT1 siRNA 3 was efficient in knocking down SIRT1. Total extracellular signal–regulated kinase (T-ERK) remained unchanged. Values are presented as mean ± SD of n = 3. ***P < 0.001 vs. siRNA-NC by one-way ANOVA with Tukey test. C: Primary DRG neurons were cultured with siRNA-NC or SIRT1-siRNA3 for 30 h and then cultured in normal (10 mmol/L glucose) or HG (45 mmol/L) medium for 24 h. Western blot and its quantification results demonstrated that SIRT1 expression was obviously decreased after treatment with SIRT1 siRNA 3 (lanes 3–6). The expressions of SIRT1 and PGC-1α in HG-cultured (lane 2) DRG neurons were decreased while p-NF-κB was increased compared with that in normal medium (lane 1). DW14006 did not impact the expressions of p-NF-κB and PGC-1α after treatment with SIRT1 siRNA 3 (lanes 4–6) in HG-cultured DRG neurons compared with that in HG-cultured DRG neurons without DW14006 (lane 3). D: Quantitative analyses for C. Values are presented as mean ± SD of n = 3. **P < 0.01, 2 vs. 1 by Student t test. ##P < 0.01, 3 vs. 2 by Student t test. Data were analyzed by Student t test (two groups) or one-way ANOVA with Tukey test (three or more than three groups). ns, not significant.

Figure 6

DW14006 had no effects on the expressions of p-NF-κB and PGC-1α in HG-cultured DRG neurons treated with SIRT1 siRNA. A and B: Western blot and its quantification results demonstrated that SIRT1 siRNA 3 was efficient in knocking down SIRT1. Total extracellular signal–regulated kinase (T-ERK) remained unchanged. Values are presented as mean ± SD of n = 3. ***P < 0.001 vs. siRNA-NC by one-way ANOVA with Tukey test. C: Primary DRG neurons were cultured with siRNA-NC or SIRT1-siRNA3 for 30 h and then cultured in normal (10 mmol/L glucose) or HG (45 mmol/L) medium for 24 h. Western blot and its quantification results demonstrated that SIRT1 expression was obviously decreased after treatment with SIRT1 siRNA 3 (lanes 3–6). The expressions of SIRT1 and PGC-1α in HG-cultured (lane 2) DRG neurons were decreased while p-NF-κB was increased compared with that in normal medium (lane 1). DW14006 did not impact the expressions of p-NF-κB and PGC-1α after treatment with SIRT1 siRNA 3 (lanes 4–6) in HG-cultured DRG neurons compared with that in HG-cultured DRG neurons without DW14006 (lane 3). D: Quantitative analyses for C. Values are presented as mean ± SD of n = 3. **P < 0.01, 2 vs. 1 by Student t test. ##P < 0.01, 3 vs. 2 by Student t test. Data were analyzed by Student t test (two groups) or one-way ANOVA with Tukey test (three or more than three groups). ns, not significant.

DW14006 Downregulated the Akt/Mammalian Target of Rapamycin Pathway in DRG Tissue From Diabetic Mice

Given that mammalian target of rapamycin (mTOR) is a target of AMPK and participates in protein synthesis by a high energy-consuming process (37), we detected the potential of DW14006 in regulating mTOR signaling.

As indicated in Supplementary Fig. 8, levels of phosphorylated Akt (p-Akt) (Thr308) and phosphorylated mTOR (p-mTOR) (Ser2448) expressions were increased in diabetic mice compared with those in control mice, and DW14006 treatment decreased p-Akt and p-mTOR expressions in DRG homogenate from diabetic mice.

DW14006 Reduced Oxidative Stress in Diabetic Mice by Activating the AMPKα/Nuclear Factor Erythroid-2–Related Factor 2 Pathway

Given that oxidative stress can be initiated by high concentrations of glucose in diabetic mice (12,13), we examined the potential effect of DW14006 treatment on oxidative stress in DRG tissue and serum of diabetic mice.

DW14006 Reduced Oxidative Stress Levels

Given that superoxide is a vital component of ROS (12), we detected superoxide level (MitoSOX) in DRG neurons from mice. As indicated in Fig. 7A, B, and G, the superoxide level was upregulated in DRG neurons from diabetic mice, and DW14006 treatment downregulated such a level. Besides, 8-hydroxy-2deoxyguanosine is a biomarker of mtDNA oxidative damage (38), and our data also suggested that DW14006 reduced 8-hydroxy-2deoxyguanosine fluorescence intensity in DRG neurons from diabetic mice by activating AMPKα, which was verified by AMPKα knockdown assay in STZ+AAV8-AMPKα-RNAi mice (Supplementary Fig. 5A–E). Similarly, SOD, GSH, and MDA (product of lipid peroxidation) were also assayed to evaluate the level of oxidative stress (39). As indicated in Supplementary Fig. 5F–H, DW14006 increased SOD activity and GSH level, while it decreased MDA levels in serum from diabetic mice by activating AMPKα as verified by an AMPKα knockdown assay in STZ+AAV8-AMPKα-RNAi mice.

Figure 7

DW14006 treatment reduced superoxide level and promoted Nrf2 nucleus translocation in DRG neurons from diabetic mice by activating AMPKα. A and B: MitoSOX red mitochondrial superoxide indicator (red) and mitochondria tracker fluorescence dye (green) were used to measure superoxide in the mitochondria of live cells. CE: Representative immunofluorescent images showing neuron-specific β-tubulin isotype III (green), Nrf2 (red), and DAPI (nucleus, blue) staining in DRG sections. Scale bars = 50 μm. F: Quantification of nucleus Nrf2 fluorescence intensity for E. Values are presented as mean ± SEM of n = 8/group. G: Quantification of MitoSOX intensity for A and B. Values are presented as mean ± SEM of n = 3 replicate cultures. H: Quantification of nucleus Nrf2 fluorescence intensity for C and D. Values are presented as mean ± SEM of n = 8/group. ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

Figure 7

DW14006 treatment reduced superoxide level and promoted Nrf2 nucleus translocation in DRG neurons from diabetic mice by activating AMPKα. A and B: MitoSOX red mitochondrial superoxide indicator (red) and mitochondria tracker fluorescence dye (green) were used to measure superoxide in the mitochondria of live cells. CE: Representative immunofluorescent images showing neuron-specific β-tubulin isotype III (green), Nrf2 (red), and DAPI (nucleus, blue) staining in DRG sections. Scale bars = 50 μm. F: Quantification of nucleus Nrf2 fluorescence intensity for E. Values are presented as mean ± SEM of n = 8/group. G: Quantification of MitoSOX intensity for A and B. Values are presented as mean ± SEM of n = 3 replicate cultures. H: Quantification of nucleus Nrf2 fluorescence intensity for C and D. Values are presented as mean ± SEM of n = 8/group. ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

DW14006 Activated Nuclear Factor Erythroid-2–Related Factor 2 Signaling

In oxidative stress, nuclear factor erythroid-2–related factor 2 (Nrf2) is released and translocated into the nucleus, thus triggering the expressions of antioxidant response element–mediated antioxidant enzymes and the cystine/glutamate transporters related to glutathione biosynthesis (40). Thus, we investigated the potential of DW14006 in regulating Nrf2 nuclear translocation in response to its antioxidation effect in DRG neurons. As shown in Fig. 7C, D, and H, Nrf2 fluorescence intensity in the nucleus of DRG neurons was decreased from diabetic mice but increased from DW14006-treated diabetic mice. Notably, DW14006 administration failed to affect Nrf2 fluorescence intensity in the nucleus of DRG tissues from DW14006-treated STZ+AAV8-AMPKα-RNAi mice (Fig. 7E and F).

DW14006 Suppressed Inflammation in Diabetic Mice Through AMPKα/NF-κB Signaling

Given the close linkage of inflammation to DPN, we detected the potential regulation of DW14006 against inflammation by ELISA, Western blot, and immunofluorescence assays.

DW14006 Reduced Proinflammatory Cytokines

Western blot results indicated that the protein levels of TNF-α and inducible nitric oxide synthase (iNOS) in splenic tissues were increased from diabetic mice and decreased from DW14006-treated diabetic mice (Fig. 8A and C). Besides, ELISA results also demonstrated that DW14006 treatment obviously suppressed the levels of TNF-α and IL-6 (Fig. 8E). Notably, DW14006 administration rendered no influence on TNF-α levels in serum from DW14006-treated STZ+AAV8-AMPKα-RNAi mice (Fig. 8K).

Figure 8

DW14006 treatment reduced inflammatory response in diabetic mice by activating AMPKα. A: Western blot results showing the expressions of iNOS and TNF-α in spleen from control, db/m, STZ, and db/db mice and DW14006-treated (15, 30 mg/kg) diabetic mice (STZ+15, 30 mg/kg, db/db+15, 30 mg/kg). B: Western blot results showing the expressions of p-IκBα and IκBα in DRG from control or db/m mice, STZ or db/db mice, and DW14006-treated (15, 30 mg/kg) diabetic mice (STZ+15, 30 mg/kg, db/db+15, 30 mg/kg). C: Quantification of expressions of iNOS and TNF-α, normalized to β-actin for A. Values are presented as mean ± SD of n = 3/group. D: Quantification of expression of p-IκBα, normalized to total IκBα expression level for B. Values are presented as mean ± SD of n = 3/group. E: ELISA results indicated that DW14006 treatment (15, 30 mg/kg) decreased TNF-α and IL-6 levels in serum from type 1 (STZ) and 2 (db/db) diabetic mice. Values are presented as mean ± SEM of n = 10/group. F and G: Representative immunofluorescent images showing neuron-specific β-tubulin isotype III (neuron, green), NF-κB (NF-κB antibody, red), and DAPI (nucleus, blue) staining in DRG sections from control or db/m mice, STZ or db/db mice, and DW14006-treated (15, 30 mg/kg) diabetic mice (STZ+15, 30 mg/kg, db/db+15, 30 mg/kg). Scale bar = 50 μm. H: Quantification of nucleus NF-κB fluorescence intensity for F and G. Values are presented as mean ± SEM of n = 8/group. I: Representative immunofluorescent images showing neuron-specific β-tubulin isotype III (neuron, green), NF-κB (NF-κB antibody, red), and DAPI (nucleus, blue) staining in DRG sections from AAV8-injected STZ mice (STZ+AAV8-NC, STZ+AAV8-AMPKα-RNAi and STZ+AAV8-AMPKα-RNAi+DW14006). J: Quantification of nucleus NF-κB fluorescence intensity for I. Values are presented as mean ± SEM of n = 8/group. K: DW14006 exerted no effect on TNF-α level in STZ+AAV8-AMPKα-RNAi mice. Values are presented as mean ± SEM of n = 10/group. L: Proposed schematic diagram illustrating the mechanisms underlying the improvement of DW14006 on DPN-like pathology. *P < 0.05, ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

Figure 8

DW14006 treatment reduced inflammatory response in diabetic mice by activating AMPKα. A: Western blot results showing the expressions of iNOS and TNF-α in spleen from control, db/m, STZ, and db/db mice and DW14006-treated (15, 30 mg/kg) diabetic mice (STZ+15, 30 mg/kg, db/db+15, 30 mg/kg). B: Western blot results showing the expressions of p-IκBα and IκBα in DRG from control or db/m mice, STZ or db/db mice, and DW14006-treated (15, 30 mg/kg) diabetic mice (STZ+15, 30 mg/kg, db/db+15, 30 mg/kg). C: Quantification of expressions of iNOS and TNF-α, normalized to β-actin for A. Values are presented as mean ± SD of n = 3/group. D: Quantification of expression of p-IκBα, normalized to total IκBα expression level for B. Values are presented as mean ± SD of n = 3/group. E: ELISA results indicated that DW14006 treatment (15, 30 mg/kg) decreased TNF-α and IL-6 levels in serum from type 1 (STZ) and 2 (db/db) diabetic mice. Values are presented as mean ± SEM of n = 10/group. F and G: Representative immunofluorescent images showing neuron-specific β-tubulin isotype III (neuron, green), NF-κB (NF-κB antibody, red), and DAPI (nucleus, blue) staining in DRG sections from control or db/m mice, STZ or db/db mice, and DW14006-treated (15, 30 mg/kg) diabetic mice (STZ+15, 30 mg/kg, db/db+15, 30 mg/kg). Scale bar = 50 μm. H: Quantification of nucleus NF-κB fluorescence intensity for F and G. Values are presented as mean ± SEM of n = 8/group. I: Representative immunofluorescent images showing neuron-specific β-tubulin isotype III (neuron, green), NF-κB (NF-κB antibody, red), and DAPI (nucleus, blue) staining in DRG sections from AAV8-injected STZ mice (STZ+AAV8-NC, STZ+AAV8-AMPKα-RNAi and STZ+AAV8-AMPKα-RNAi+DW14006). J: Quantification of nucleus NF-κB fluorescence intensity for I. Values are presented as mean ± SEM of n = 8/group. K: DW14006 exerted no effect on TNF-α level in STZ+AAV8-AMPKα-RNAi mice. Values are presented as mean ± SEM of n = 10/group. L: Proposed schematic diagram illustrating the mechanisms underlying the improvement of DW14006 on DPN-like pathology. *P < 0.05, ***P < 0.001, STZ or db/db mice vs. control or db/m mice by Student t test. #P < 0.05, ##P < 0.01, ###P < 0.001, DW14006-treated STZ or db/db mice vs. STZ or db/db mice by one-way ANOVA with Dunnett post hoc test.

DW14006 Suppressed Inflammation Through AMPKα/NF-κB Signaling

Since activation of inhibitor of κB (IκB) α via phosphorylation results in the release and nuclear translocation of active NF-κB, leading to upregulation of proinflammatory genes (41), we detected IκBα phosphorylation in DRG homogenate from mice. As indicated in Fig. 8B and D, phosphorylated IκBα (p-IκBα) levels were increased in diabetic mice and decreased in DW14006-treated diabetic mice, thereby suggesting that DW14006 treatment suppressed IκBα phosphorylation.

Next, we inspected the nuclear translocation of NF-κB in DRG tissues from mice. As indicated in Fig. 8F–H, the red fluorescence intensity (NF-κB antibody) in the nucleus was obviously increased in diabetic mice and decreased in DW14006-treated diabetic mice. Notably, DW14006 treatment gave no influence on the nucleus NF-κB fluorescence intensity in DRG tissue from DW14006-treated STZ+AAV8-AMPKα-RNAi mice (Fig. 8I and J).

DPN is a long-term complication of diabetes with a complicated pathogenesis. Several indirect AMPK activators were recently reported to improve DPN, but they activated AMPK involving inhibition of mitochondrial respiration and further the reduction of mitochondrial ATP synthesis (7,16). ATP is necessary for neurite outgrowth of DRG neurons, and its abnormality in mtBEs is highly responsible for DPN etiology (12,18). DW14006 as a direct AMPKα activator improved mitochondrial function and increased mitochondrial ATP generation, implying that DW14006 might overcome the potential deficiency from indirect AMPK activators.

In addition, we determined that DW14006 promoted neurite outgrowth of DRG by regulating AMPKα/SIRT1/PGC-1α signaling axis. AMPK activation enhances SIRT1 activity by elevation of NAD+ (42), and SIRT1 catalyzes PGC-1α deacetylation (43) that is highly related to mtBE. These facts have thus highlighted the potency of AMPKα-mediated signaling in neurite outgrowth of DRGs. Moreover, diabetic complications are highly correlated with vascular disease, which is also a major cause of diabetic neuropathy (44). DW14006 treatment efficiently improved peripheral nerve tissue perfusion and blood flow velocity accompanied by nerve function improvement in diabetic mice. All results have strongly supported that activating AMPKα should be a promising therapeutic strategy for DPN.

Hyperglycemia-induced mitochondrial superoxide overproduction leads to oxidative stress, and the vast majority of cellular ROS can be traced back to the mitochondria. SIRT1 as a protein deacetylase is recognized as an effective regulator for preventing cells from oxidative stress damage. SIRT1 activation increases Nrf2 expression and its downstream genes (45) and inhibits NF-κB signaling accompanied by enhancing oxidative metabolism (46). DW14006 suppressed antioxidative stress by enhancing mitochondrial respiration and the AMPKα/SIRT1/Nrf2 signaling axis, thus providing a new strategy for designing reagents against DPN. Additionally, we detected the effects of DW14006 in HG-cultured DRG neurons treated with SIRT1 siRNA (Fig. 6). The results indicated that SIRT1 knockdown phenocopied the effects of AMPK knockdown and has highlighted the central node of SIRT1 in connecting AMPK to improving mitochondrial function and decreasing inflammation.

Notably, DW14006 treatment rendered no impact on the body weight of STZ mice but decreased the body weight of db/db mice. Here, we tentatively ascribed this finding to the lower level of total cholesterol in DW14006-treated db/db mice (Supplementary Fig. 2), and we suggested that some of the improvements of DW14006 in db/db mice may benefit from the reduction of lipid accumulation. In addition, the result that DW14006 treatment had no effects on blood glucose levels in diabetic mice highly verified the benefit of DW14006 on DPN were independent of blood glucose regulation. Interestingly, we also determined that STZ mice showed a hypersensitivity in mechanical stimulus at early stage (first 2 weeks) of diabetes but exhibited a decreased sensitivity in mechanical stimulus after 4 weeks of STZ injection. On the basis of the published reports (17,47,48), we speculated that hypersensitivity seemed to be an early feature and sensory repression and loss were advanced features of diabetic mice. Moreover, DW14006 was also determined to suppress protein synthesis involving downregulation of mTOR signaling to maintain energy homeostasis during cellular energy stress in diabetic status (Supplementary Fig. 8). To our knowledge, our work might be the first report on the cross talk between direct AMPKα activation and DPN regulation.

Finally, DPN improvement was evaluated by MNCV and sensory sensitivity assays in the current work, although these assays are very potent for DPN study (28,49) according to the Diabetes Complications Consortium guidelines, sensory nerve conduction velocity (SNVC)-related assays should be of high complementation to our work.

As summarized in Fig. 8L, DW14006 as a direct AMPKα activator ameliorated DPN-like pathology in diabetic mice by counteracting multiple risk factors with SIRT1 as a central node in connecting AMPKα to regulate mitochondrial function. It enhanced mitochondrial function through the AMPKα/SIRT1/PGC-1α pathway, repressed inflammation via AMPKα/NF-κB signaling, and suppressed oxidative stress via the AMPKα/Nrf2 pathway.

This article contains supplementary material online at https://doi.org/10.2337/figshare.12583115.

Xu Xu, W.W., and Z.W. contributed equally.

Acknowledgments. The authors thank Linnan Qian (Nanjing University of Chinese Medicine) for expert technical assistance. The authors thank Jiaxun Nie, Xue Lu, and Rui Guo (Nanjing University of Chinese Medicine) for offering guides on immunohistochemistry staining and MNCV test.

Funding. This work was supported by National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (2018ZX09711002), the National Natural Science Foundation for Young Scientists of China (81703806), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1600), the Open Project Program of Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica (JKLPSE201801), and the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions(Integration of Chinese and Western Medicine).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Xu Xu and X.S. designed the study. W.W., Z.W., W.D., and J.Z. synthetized DW14006. Xu Xu, J.L., Xi. Xu, J.X., J.Y., X.Z., Y.L., X.H., and J.W. performed the animal experiments. Xu Xu analyzed and interpreted data. Xu Xu wrote the manuscript. X.S. reviewed the manuscript. All authors approved the manuscript. J.Z. and X.S. are the guarantors of this work and, as such, had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

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