Strengthening monocarboxylate transporters by adiponectin receptor agonist ameliorates diabetic peripheral neuropathy

Abstract

Background: Disruption of energy support to peripheral nerves leads to dysfunction and degeneration of both neuronal axons and Schwann cells (SCs).

Methods: We evaluated the effects of the adiponectin receptor (AdipoR) agonist, AdipoRon on diabetic peripheral neuropathy (DPN) using db/db mice, murine ND7/23 cells, and human SCs.

Results: AdipoRon improved sensorimotor function and restored nerve phenotypes in the sciatic nerve and paw skin of db/db mice by reducing systemic oxidative stress and insulin resistance. AdipoRon restored impaired oxidative stress response, apoptosis, and autophagy activity through increased AdipoR1/R2-intracellular Ca⁺⁺-CaMKKβ expression as well as LKB1/AMPK-PPARα/PGC-1α/Nrf2 phosphorylation. It also decreased mTOR phosphorylation, which is related to the preservation of mitochondria in axons and SCs, and improved MCT1/2/4 expression and lactate and ATP/AMP levels in the sciatic nerve of db/db mice. In ND7/23 cells and SCs, AdipoRon decreased oxidative stress and apoptosis while increasing autophagy by suppressing high glucose- and palmitate-induced cellular and mitochondrial oxidative stress, activating the same signaling as in the sciatic nerve. These protective changes were induced by providing energy substrate, lactate and ATP, through the increased expression of MCT1/2/4, facilitating metabolic communication between neurons and SCs.

Conclusion: AdipoRon may play an important role in preventing DPN by ameliorating oxidative stress, apoptosis, and autophagy, strengthening metabolic support for neurons and SCs under diabetic conditions.

Keywords: Adiponectin; Diabetic peripheral neuropathy; Glucolipotoxicity; Monocarboxylate transporter.

Fig. 1

Effects of AdipoRon on nerve function and morphology in diabetic db/db and non-diabetic db/m mice. (A) Tactile thresholds, motor nerve conduction latency (MNCL), and action potential amplitude, along with electron microscopy results showing axonal area, unmyelinated nerve area, axonal diameter, and G-ratio in the sciatic nerve of diabetic db/db and non-diabetic db/m mice with or without AdipoRon treatment, are shown with quantitative analyses (n = 6). Red arrows indicate aberrant structures such as redundant myelin folding, infolding of the myelin sheaths, and degenerated axons (scale bars: 2 μm). (B) Immunofluorescence (IF) staining of collagen IV (red, scale bars: 10 μm), 8-OH-dG (green, 10 μm), and total Nrf2 (green, 10 μm) was performed individually. Double IF staining was performed for β3-tubulin (red, 20 μm) with either TUNEL or LC3 (green, 20 μm) to confirm the neuronal localization of apoptotic and autophagic signals. DAPI (blue) was used for nuclear counterstaining, and representative images with their enlarged views from the rectangular areas and corresponding quantitative analyses are shown (n = 6). (C) Additional experiments in paw skin included Masson’s Trichrome (MT) staining (scale bars: 100 μm) to assess fibrosis. Immunofluorescence (IF) staining included the following double combinations: SOX10 (green, Schwann cell marker, indicated by white open arrows) and PGP9.5 (red, nerve fiber marker, indicated by red open arrowheads) (scale bars: 20 μm); collagen IV (red) and 8-OH-dG (green) (scale bars: 20 μm); and β3-tubulin (red, mature neuron marker) with either TUNEL or LC3 (green) (scale bars: 10 μm). DAPI (blue) was used for nuclear counterstaining, and representative images with corresponding analyses are shown (n = 6). All analyses were performed in db/db and db/m mice with or without AdipoRon treatment. Data are presented as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, and #P < 0.001 compared with other groups

Fig. 1

Effects of AdipoRon on nerve function and morphology in diabetic db/db and non-diabetic db/m mice. (A) Tactile thresholds, motor nerve conduction latency (MNCL), and action potential amplitude, along with electron microscopy results showing axonal area, unmyelinated nerve area, axonal diameter, and G-ratio in the sciatic nerve of diabetic db/db and non-diabetic db/m mice with or without AdipoRon treatment, are shown with quantitative analyses (n = 6). Red arrows indicate aberrant structures such as redundant myelin folding, infolding of the myelin sheaths, and degenerated axons (scale bars: 2 μm). (B) Immunofluorescence (IF) staining of collagen IV (red, scale bars: 10 μm), 8-OH-dG (green, 10 μm), and total Nrf2 (green, 10 μm) was performed individually. Double IF staining was performed for β3-tubulin (red, 20 μm) with either TUNEL or LC3 (green, 20 μm) to confirm the neuronal localization of apoptotic and autophagic signals. DAPI (blue) was used for nuclear counterstaining, and representative images with their enlarged views from the rectangular areas and corresponding quantitative analyses are shown (n = 6). (C) Additional experiments in paw skin included Masson’s Trichrome (MT) staining (scale bars: 100 μm) to assess fibrosis. Immunofluorescence (IF) staining included the following double combinations: SOX10 (green, Schwann cell marker, indicated by white open arrows) and PGP9.5 (red, nerve fiber marker, indicated by red open arrowheads) (scale bars: 20 μm); collagen IV (red) and 8-OH-dG (green) (scale bars: 20 μm); and β3-tubulin (red, mature neuron marker) with either TUNEL or LC3 (green) (scale bars: 10 μm). DAPI (blue) was used for nuclear counterstaining, and representative images with corresponding analyses are shown (n = 6). All analyses were performed in db/db and db/m mice with or without AdipoRon treatment. Data are presented as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, and #P < 0.001 compared with other groups

Fig. 1

Effects of AdipoRon on nerve function and morphology in diabetic db/db and non-diabetic db/m mice. (A) Tactile thresholds, motor nerve conduction latency (MNCL), and action potential amplitude, along with electron microscopy results showing axonal area, unmyelinated nerve area, axonal diameter, and G-ratio in the sciatic nerve of diabetic db/db and non-diabetic db/m mice with or without AdipoRon treatment, are shown with quantitative analyses (n = 6). Red arrows indicate aberrant structures such as redundant myelin folding, infolding of the myelin sheaths, and degenerated axons (scale bars: 2 μm). (B) Immunofluorescence (IF) staining of collagen IV (red, scale bars: 10 μm), 8-OH-dG (green, 10 μm), and total Nrf2 (green, 10 μm) was performed individually. Double IF staining was performed for β3-tubulin (red, 20 μm) with either TUNEL or LC3 (green, 20 μm) to confirm the neuronal localization of apoptotic and autophagic signals. DAPI (blue) was used for nuclear counterstaining, and representative images with their enlarged views from the rectangular areas and corresponding quantitative analyses are shown (n = 6). (C) Additional experiments in paw skin included Masson’s Trichrome (MT) staining (scale bars: 100 μm) to assess fibrosis. Immunofluorescence (IF) staining included the following double combinations: SOX10 (green, Schwann cell marker, indicated by white open arrows) and PGP9.5 (red, nerve fiber marker, indicated by red open arrowheads) (scale bars: 20 μm); collagen IV (red) and 8-OH-dG (green) (scale bars: 20 μm); and β3-tubulin (red, mature neuron marker) with either TUNEL or LC3 (green) (scale bars: 10 μm). DAPI (blue) was used for nuclear counterstaining, and representative images with corresponding analyses are shown (n = 6). All analyses were performed in db/db and db/m mice with or without AdipoRon treatment. Data are presented as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, and #P < 0.001 compared with other groups

Fig. 2

Changes in the expressions of adiponectin receptors and associated downstream signaling pathways in the sciatic nerve of diabetic db/db and non-diabetic db/m mice with or without AdipoRon treatment. (A) Representative western blot images of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PPARα, PGC-1α, p-Nrf2/total Nrf2, MCT1, MCT2, MCT4, GAPDH, and Bax/Bcl-2 and LC3-II/LC3-I ratios in the sciatic nerve are shown with quantitative analyses (n = 3). (B) Representative images of triple IF staining for MCT1, MCT2, and MCT4 (green), NF (white, axonal marker), and MBP (red, myelin marker) in the sciatic nerve of db/db and db/m mice with or without AdipoRon are shown with corresponding quantitative analyses (n = 6). (C) Expression levels of LDHA and LDHB in the sciatic nerve after AdipoRon treatment, evaluated by western blot (n = 3) and IF analysis and their enlarged images from the rectangular areas, respectively, are shown with quantitative analyses (n = 6). (D) Quantitative measurements of lactate, AMP, and ATP levels, including the ATP/AMP ratio, in the sciatic nerve of db/db mice with or without AdipoRon treatment (n = 4). (E) Representative electron microscopy images of the sciatic nerve (x 5,000, scale bars: 2 μm) in db/db and db/m mice with or without AdipoRon treatment and their enlarged images from the rectangular areas, respectively. Quantitative analyses of mitochondrial number in myelinated axons (n = 10) and Schwann cells (n = 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.001 compared with other groups. Scale bars in (B) represent 2 μm, and in (C), 10 μm

Fig. 2

Changes in the expressions of adiponectin receptors and associated downstream signaling pathways in the sciatic nerve of diabetic db/db and non-diabetic db/m mice with or without AdipoRon treatment. (A) Representative western blot images of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PPARα, PGC-1α, p-Nrf2/total Nrf2, MCT1, MCT2, MCT4, GAPDH, and Bax/Bcl-2 and LC3-II/LC3-I ratios in the sciatic nerve are shown with quantitative analyses (n = 3). (B) Representative images of triple IF staining for MCT1, MCT2, and MCT4 (green), NF (white, axonal marker), and MBP (red, myelin marker) in the sciatic nerve of db/db and db/m mice with or without AdipoRon are shown with corresponding quantitative analyses (n = 6). (C) Expression levels of LDHA and LDHB in the sciatic nerve after AdipoRon treatment, evaluated by western blot (n = 3) and IF analysis and their enlarged images from the rectangular areas, respectively, are shown with quantitative analyses (n = 6). (D) Quantitative measurements of lactate, AMP, and ATP levels, including the ATP/AMP ratio, in the sciatic nerve of db/db mice with or without AdipoRon treatment (n = 4). (E) Representative electron microscopy images of the sciatic nerve (x 5,000, scale bars: 2 μm) in db/db and db/m mice with or without AdipoRon treatment and their enlarged images from the rectangular areas, respectively. Quantitative analyses of mitochondrial number in myelinated axons (n = 10) and Schwann cells (n = 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.001 compared with other groups. Scale bars in (B) represent 2 μm, and in (C), 10 μm

Fig. 2

Changes in the expressions of adiponectin receptors and associated downstream signaling pathways in the sciatic nerve of diabetic db/db and non-diabetic db/m mice with or without AdipoRon treatment. (A) Representative western blot images of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PPARα, PGC-1α, p-Nrf2/total Nrf2, MCT1, MCT2, MCT4, GAPDH, and Bax/Bcl-2 and LC3-II/LC3-I ratios in the sciatic nerve are shown with quantitative analyses (n = 3). (B) Representative images of triple IF staining for MCT1, MCT2, and MCT4 (green), NF (white, axonal marker), and MBP (red, myelin marker) in the sciatic nerve of db/db and db/m mice with or without AdipoRon are shown with corresponding quantitative analyses (n = 6). (C) Expression levels of LDHA and LDHB in the sciatic nerve after AdipoRon treatment, evaluated by western blot (n = 3) and IF analysis and their enlarged images from the rectangular areas, respectively, are shown with quantitative analyses (n = 6). (D) Quantitative measurements of lactate, AMP, and ATP levels, including the ATP/AMP ratio, in the sciatic nerve of db/db mice with or without AdipoRon treatment (n = 4). (E) Representative electron microscopy images of the sciatic nerve (x 5,000, scale bars: 2 μm) in db/db and db/m mice with or without AdipoRon treatment and their enlarged images from the rectangular areas, respectively. Quantitative analyses of mitochondrial number in myelinated axons (n = 10) and Schwann cells (n = 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.001 compared with other groups. Scale bars in (B) represent 2 μm, and in (C), 10 μm

Fig. 2

Changes in the expressions of adiponectin receptors and associated downstream signaling pathways in the sciatic nerve of diabetic db/db and non-diabetic db/m mice with or without AdipoRon treatment. (A) Representative western blot images of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PPARα, PGC-1α, p-Nrf2/total Nrf2, MCT1, MCT2, MCT4, GAPDH, and Bax/Bcl-2 and LC3-II/LC3-I ratios in the sciatic nerve are shown with quantitative analyses (n = 3). (B) Representative images of triple IF staining for MCT1, MCT2, and MCT4 (green), NF (white, axonal marker), and MBP (red, myelin marker) in the sciatic nerve of db/db and db/m mice with or without AdipoRon are shown with corresponding quantitative analyses (n = 6). (C) Expression levels of LDHA and LDHB in the sciatic nerve after AdipoRon treatment, evaluated by western blot (n = 3) and IF analysis and their enlarged images from the rectangular areas, respectively, are shown with quantitative analyses (n = 6). (D) Quantitative measurements of lactate, AMP, and ATP levels, including the ATP/AMP ratio, in the sciatic nerve of db/db mice with or without AdipoRon treatment (n = 4). (E) Representative electron microscopy images of the sciatic nerve (x 5,000, scale bars: 2 μm) in db/db and db/m mice with or without AdipoRon treatment and their enlarged images from the rectangular areas, respectively. Quantitative analyses of mitochondrial number in myelinated axons (n = 10) and Schwann cells (n = 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.001 compared with other groups. Scale bars in (B) represent 2 μm, and in (C), 10 μm

Fig. 3

AdipoRon modulates AdipoR1 and AdipoR2 expression in human Schwann cells (HSCs). Human Schwann cells (HSCs) were transfected with adipoR1 or adipoR2 siRNA and cultured in high glucose and palmitate medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin B. Quantitative analyses of protein expression levels are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with MBP (red, myelin marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and IF images (n = 6) for LDHA and LDHB expression are shown with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, including the ATP/AMP ratio, are shown (n = 5). (E) Representative double immunofluorescence staining images of MCT4 (green) and MitoSOX (red) in HSCs under different conditions (n = 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C and E) represent 20 μm

Fig. 3

AdipoRon modulates AdipoR1 and AdipoR2 expression in human Schwann cells (HSCs). Human Schwann cells (HSCs) were transfected with adipoR1 or adipoR2 siRNA and cultured in high glucose and palmitate medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin B. Quantitative analyses of protein expression levels are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with MBP (red, myelin marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and IF images (n = 6) for LDHA and LDHB expression are shown with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, including the ATP/AMP ratio, are shown (n = 5). (E) Representative double immunofluorescence staining images of MCT4 (green) and MitoSOX (red) in HSCs under different conditions (n = 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C and E) represent 20 μm

Fig. 3

AdipoRon modulates AdipoR1 and AdipoR2 expression in human Schwann cells (HSCs). Human Schwann cells (HSCs) were transfected with adipoR1 or adipoR2 siRNA and cultured in high glucose and palmitate medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin B. Quantitative analyses of protein expression levels are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with MBP (red, myelin marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and IF images (n = 6) for LDHA and LDHB expression are shown with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, including the ATP/AMP ratio, are shown (n = 5). (E) Representative double immunofluorescence staining images of MCT4 (green) and MitoSOX (red) in HSCs under different conditions (n = 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C and E) represent 20 μm

Fig. 4

Effect of AdipoRon on signaling pathways in ND7/23 cells (murine DRG neuronal cell). ND7/23 cells were transfected with adipoR1 or adipoR2 siRNA and cultured in low- or high-glucose (HG) and palmitate (PA) medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, Bax/Bcl-2, LC3II/LC3I ratios, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin (B). Quantitative analyses of protein expression levels in each group are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with NF (white, axonal marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and double IF images (n = 6) for LDHA (red) and LDHB (green) expression, with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, and ATP/AMP ratio (n = 5). (E) Representative sections stained with Fluo-4AM (green, intracellular Ca⁺⁺), DHE (red, cellular oxidative stress), MitoSox (red, mitochondrial oxidative stress), TUNEL (green, apoptosis), and LC3 (green, autophagy). Quantitative analyses of positive areas and density are shown for each group. Data are presented as the mean ± SD (n = 6). (F) Representative double IF staining images of MCT1, MCT2, MCT4 (green) and MitoSOX (red) in ND7/23 cells under different conditions (n = 6). (G) Quantitative measurements of intracellular lactate, ATP and AMP concentrations and ATP/AMP ratios in SCs and ND7/23 cells in low glucose (LG), high glucose and palmitate (HG + PA), and high glucose and palmitate treated with adiopoRon (HG + PA + adipoR), respectively (n = 5). *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C, E and F) represent 5 μm

Fig. 4

Effect of AdipoRon on signaling pathways in ND7/23 cells (murine DRG neuronal cell). ND7/23 cells were transfected with adipoR1 or adipoR2 siRNA and cultured in low- or high-glucose (HG) and palmitate (PA) medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, Bax/Bcl-2, LC3II/LC3I ratios, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin (B). Quantitative analyses of protein expression levels in each group are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with NF (white, axonal marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and double IF images (n = 6) for LDHA (red) and LDHB (green) expression, with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, and ATP/AMP ratio (n = 5). (E) Representative sections stained with Fluo-4AM (green, intracellular Ca⁺⁺), DHE (red, cellular oxidative stress), MitoSox (red, mitochondrial oxidative stress), TUNEL (green, apoptosis), and LC3 (green, autophagy). Quantitative analyses of positive areas and density are shown for each group. Data are presented as the mean ± SD (n = 6). (F) Representative double IF staining images of MCT1, MCT2, MCT4 (green) and MitoSOX (red) in ND7/23 cells under different conditions (n = 6). (G) Quantitative measurements of intracellular lactate, ATP and AMP concentrations and ATP/AMP ratios in SCs and ND7/23 cells in low glucose (LG), high glucose and palmitate (HG + PA), and high glucose and palmitate treated with adiopoRon (HG + PA + adipoR), respectively (n = 5). *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C, E and F) represent 5 μm

Fig. 4

Effect of AdipoRon on signaling pathways in ND7/23 cells (murine DRG neuronal cell). ND7/23 cells were transfected with adipoR1 or adipoR2 siRNA and cultured in low- or high-glucose (HG) and palmitate (PA) medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, Bax/Bcl-2, LC3II/LC3I ratios, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin (B). Quantitative analyses of protein expression levels in each group are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with NF (white, axonal marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and double IF images (n = 6) for LDHA (red) and LDHB (green) expression, with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, and ATP/AMP ratio (n = 5). (E) Representative sections stained with Fluo-4AM (green, intracellular Ca⁺⁺), DHE (red, cellular oxidative stress), MitoSox (red, mitochondrial oxidative stress), TUNEL (green, apoptosis), and LC3 (green, autophagy). Quantitative analyses of positive areas and density are shown for each group. Data are presented as the mean ± SD (n = 6). (F) Representative double IF staining images of MCT1, MCT2, MCT4 (green) and MitoSOX (red) in ND7/23 cells under different conditions (n = 6). (G) Quantitative measurements of intracellular lactate, ATP and AMP concentrations and ATP/AMP ratios in SCs and ND7/23 cells in low glucose (LG), high glucose and palmitate (HG + PA), and high glucose and palmitate treated with adiopoRon (HG + PA + adipoR), respectively (n = 5). *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C, E and F) represent 5 μm

Fig. 4

Effect of AdipoRon on signaling pathways in ND7/23 cells (murine DRG neuronal cell). ND7/23 cells were transfected with adipoR1 or adipoR2 siRNA and cultured in low- or high-glucose (HG) and palmitate (PA) medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, Bax/Bcl-2, LC3II/LC3I ratios, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin (B). Quantitative analyses of protein expression levels in each group are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with NF (white, axonal marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and double IF images (n = 6) for LDHA (red) and LDHB (green) expression, with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, and ATP/AMP ratio (n = 5). (E) Representative sections stained with Fluo-4AM (green, intracellular Ca⁺⁺), DHE (red, cellular oxidative stress), MitoSox (red, mitochondrial oxidative stress), TUNEL (green, apoptosis), and LC3 (green, autophagy). Quantitative analyses of positive areas and density are shown for each group. Data are presented as the mean ± SD (n = 6). (F) Representative double IF staining images of MCT1, MCT2, MCT4 (green) and MitoSOX (red) in ND7/23 cells under different conditions (n = 6). (G) Quantitative measurements of intracellular lactate, ATP and AMP concentrations and ATP/AMP ratios in SCs and ND7/23 cells in low glucose (LG), high glucose and palmitate (HG + PA), and high glucose and palmitate treated with adiopoRon (HG + PA + adipoR), respectively (n = 5). *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C, E and F) represent 5 μm

Fig. 4

Effect of AdipoRon on signaling pathways in ND7/23 cells (murine DRG neuronal cell). ND7/23 cells were transfected with adipoR1 or adipoR2 siRNA and cultured in low- or high-glucose (HG) and palmitate (PA) medium, with or without AdipoRon treatment. (A) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, Bax/Bcl-2, LC3II/LC3I ratios, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin (B). Quantitative analyses of protein expression levels in each group are shown (n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with NF (white, axonal marker) are shown with corresponding quantitative analyses (n = 6). (C) Representative western blot (n = 3) and double IF images (n = 6) for LDHA (red) and LDHB (green) expression, with quantitative analyses. (D) Quantitative measurements of intracellular lactate, AMP, and ATP levels, and ATP/AMP ratio (n = 5). (E) Representative sections stained with Fluo-4AM (green, intracellular Ca⁺⁺), DHE (red, cellular oxidative stress), MitoSox (red, mitochondrial oxidative stress), TUNEL (green, apoptosis), and LC3 (green, autophagy). Quantitative analyses of positive areas and density are shown for each group. Data are presented as the mean ± SD (n = 6). (F) Representative double IF staining images of MCT1, MCT2, MCT4 (green) and MitoSOX (red) in ND7/23 cells under different conditions (n = 6). (G) Quantitative measurements of intracellular lactate, ATP and AMP concentrations and ATP/AMP ratios in SCs and ND7/23 cells in low glucose (LG), high glucose and palmitate (HG + PA), and high glucose and palmitate treated with adiopoRon (HG + PA + adipoR), respectively (n = 5). *P < 0.05, **P < 0.01, and #P < 0.001, compared with other groups. Scale bars in (B, C, E and F) represent 5 μm

Fig. 5

Mechanisms of AdipoRon in ameliorating diabetic peripheral neuropathy (DPN). AdipoRon enhances CaMKKβ/LKB1/AMPK/PPARα/PGC-1α/Nrf2 signaling and suppresses mTOR via increased AdipoR1 and AdipoR2 expression, promoting neuronal and Schwann cell survival, reducing apoptosis, and enhancing autophagy under diabetic conditions. Increased MCT1, MCT2, and MCT4 expression facilitates energy substrate supply (lactate and ATP) and metabolic communication between neurons and Schwann cells