Calcimimetic restores diabetic peripheral neuropathy by ameliorating apoptosis and improving autophagy

Decreased AMPK-eNOS bioavailability mediates the development of diabetic peripheral neuropathy (DPN) through increased apoptosis and decreased autophagy activity in relation to oxidative stress. Schwann cells are responsible for maintaining structural and functional integrity of neurons and for repairing damaged nerves. We evaluated the neuro-protective effect of cinacalcet on DPN by activating the AMPK-eNOS pathway using db/db mice and human Schwann cells (HSCs). Sciatic nerve of db/db mice was characterized by disorganized myelin, axonal shrinkage, and degeneration that were accompanied by marked fibrosis, inflammation, and apoptosis. These phenotypical alterations were significantly improved by cinacalcet treatment along with improvement in sensorimotor functional parameters. Cinacalcet demonstrated favorable effects through increased expression and activation of calcium-sensing receptor (CaSR)-CaMKKβ and phosphorylation of AMPK-eNOS signaling in diabetic sciatic nerve. Cinacalcet decreased apoptosis and increased autophagy activity in relation to decreased oxidative stress in HSCs cultured in high-glucose medium as well. This was accompanied by increased expression of the CaSR, intracellular Ca++ ([Ca++]i) levels, and CaMKKβ-LKB1-AMPK signaling pathway, resulting in the net effect of increased eNOS phosphorylation, NOx concentration, Bcl-2/Bax ratio, beclin 1, and LC3-II/LC3-I ratio. These results demonstrated that cinacalcet treatment ameliorates inflammation, apoptosis, and autophagy through increased expression of the CaSR, [Ca++]i levels and subsequent activation of CaMKKβ-LKB-1-AMPK-eNOS pathway in the sciatic nerve and HSCs under diabetic condition. Therefore, cinacalcet may play an important role in the restoration and amelioration of DPN by ameliorating apoptosis and improving autophagy.


Introduction
Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes in >50-60% of all diabetic patients, and it is also the leading cause of amputation worldwide 1,2 . The early changes in patients with DPN include accumulation of extracellular matrix proteins, inflammation, axonal degeneration, and loss of unmylelinated fibers, which cause sensorimotor conduction delays and irreversible nerve damage. It is well known that hyperglycemia plays a main role in DPN [3][4][5][6][7] with regard to the changes in oxidative-nitrosative stress, neuro-inflammation, mitochondrial dysfunction, bioenergetic crisis, and demyelination 7 .
Schwann cells (SCs) are specialized glial cells in the peripheral nervous system that are responsible for maintaining structural and functional integrity of neurons and for repairing damaged nerves 8,9 . Hyperglycemia-induced SC damages may reduce nerve conduction velocity, accelerate axonal atrophy, and impair axonal regeneration 10 .
Moreover, hyperglycemia-induced SC damages include such morphological changes as swelling and vacuolization that result in the destruction of organelles. Clearance of defective organelles constitutes the very core of the autophagy process that is an important physiological and defensive mechanism of the cell and body under such deranged metabolic conditions as nutrient or energy excess and deprivation 11 . Chronic hyperglycemia with diabetes impairs cellular autophagy and exacerbates apoptosis associated with DPN 7,12 . Autophagy promotes cell survival by sequestering senescent or damaged organelles/proteins in autophagosomes for recycling of their products 11 . Therefore, an enhancement of autophagy and a concomitant suppression of apoptosis of SCs might be the optimal strategy for the prevention and regression of DPN.
AMP-activated protein kinase (AMPK) is a master controller of cellular energy balance that activates catabolic pathways in state of energy deprivation 13 . Chronic nutrient excess state associated with prolonged diabetes triggers a switching off of AMPK, which results in impaired peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) activity and diminished mitochondrial 14 and endothelial nitric oxide synthase (eNOS) activities 15 , leading to neurodegeneration in patients with DPN. The important mode of AMPK activation relies on phosphorylation at the 172nd threonine residue of the αsubunit by upstream kinases, including Ca ++ /calmodulindependent protein kinase kinase β (CaMKKβ) and liver kinase B1 (LKB1). The LKB1 forms a complex with STRAD and MO25 in response to an elevation in AMP/ ATP ratio 16 , which phosphorylates the AMPKα subunit to trigger the AMPK pathway. CaMKKβ is an alternative upstream kinase of AMPK that responds to the change in intracellular Ca ++ ([Ca ++ ]i) concentration. Elevated [Ca ++ ]i increases the activity of AMPK, independent of the adenylate energy balance 17 .
The calcimimetic, (R)-N-(3-(3-(trifluoromethyl)phenyl) propyl)-1-(1-napthyl)ethylamine hydrochloride (cinacalcet), devised originally for the treatment of secondary hyperparathyroidism, exerts its effect by stimulating Ca ++ -sensing receptor (CaSR) mainly in the parathyroid glands 18 . Activated upon Ca ++ ions, the expression of cellular surface CaSR is crucial for maintaining a stable serum Ca ++ , which is achieved primarily through the regulation of parathyroid hormone secretion and renal Ca ++ excretion. Interestingly, the expression of CaSR has been demonstrated in the vasculature and perivascular sensory nerves 18,19 . CaSR activation by cinacalcet is known to activate CaMMK-LKB1-AMPK pathway. Activation of AMPK and LKB-1 is crucial for SC-mediated axonal maintenance while LKB deletion is responsible for axonal degeneration 20 . Moreover, the CaSR is known to modulate cell proliferation and apoptosis and coordinate oncogene expression, chemotaxis, and autophagy.
Exposed under constant metabolic stress, these axons and SCs are prone to mitochondrial dysfunction featuring derangements in [Ca ++ ]i homeostasis and associated downstream signaling that are key causal factors for the development of DPN, making it an ideal therapeutic target at the same time 21 .
To date, there is no curative therapy currently available to deter the progression of DPN; only a handful of studies have demonstrated improvements in indices of neuropathy through the activation of AMPK in cultured neurons 22 and peripheral nerves of type 1 diabetic rats 23 . On this account, we assumed that cinacalcet treatment may modulate DPN activity through the axis delineated in the previous study. Thus we investigated the protective and/ or reversal effect of cinacalcet against neural glucotoxicity through the changes in the AMPK-eNOS pathway in the sciatic nerve of db/db mice and human Schwann cells (HSCs).

Experimental animals and assessment of peripheral nerve function
All animal experiments were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee (IACUC) at College of Medicine, the Catholic University of Korea (CUMC-2014 − 0165-01). Eight-week-old male C57BLKS/J db/m and db/db mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and db/m and db/db mice were divided into four groups. Cinacalcet (10 mg/kg) mixed into standard chow diet or a regular diet was administered to db/db mice (db/db+cina; n = 8) and ageand gender-matched db/m mice (db/m+cina; n = 8) for 12 weeks starting at 8 weeks of age.
After 12 weeks of cinacalcet treatment, we performed electrophysiological and sensory threshold tests in the following order: tactile responses (a response 50% of the times the tip is applied to the hind paw) to stimulation using flexible von Frey filaments and then sciatic motor nerve conduction latency (MNCL), as described previously 15 . After the tests were completed, blood was collected from the left ventricle and the plasma was stored at −70°C for subsequent analyses, and we collected the sciatic nerves under general anesthesia with 10 mg/kg xylazine hydrochloride (Rompun; Bayer, Leuverkusen, Germany) and 30 mg/kg tiletamine plus zolazepam (Zoletil; Virbac, Carros, France). Some sciatic nerve samples were fixed in normal buffered 4% formalin for immunohistochemistry, and the others were stored in a solution for electron microscopy. We also collected the sciatic nerves in 8-week old male db/m and db/db mice for evaluation of cinacalcet effect on the recovery of DPN.

Light and electron microscopic analysis Nerve morphology
Sciatic nerve samples were fixed in 4% paraformaldehyde. Trichrome-stained nerves were used to examine the effect of cinacalcet on nerve fibrosis. Ten consecutive nerve cross-sections were photographed using a digital camera (Olympus DP11; Olympus America, Melville, NY) by an examiner who was blinded to the tissue source. Each nerve section was sampled in a serpentine pattern such that the entire nerve section was analyzed with no overlapping fields. We performed immunohistochemistry for type IV collagen (Col IV; Biodesign International, Saco, ME, USA) and 8-hydroxy-deoxyguanosine (8-OH-dG; JalCA, Fukuroi, Shizuoka, Japan), an oxidative DNA damage marker.

Electron microscopy
For transmission electron microscopy (TEM), sciatic nerves specimens were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight at 4°C. After washing in 0.1 M phosphate buffer, the specimens were post-fixed with 1% osmium tetroxide in the same buffer for 1 h. The specimens were then dehydrated using a series of graded ethanol, exchanged through acetone, and embedded in Epon 812. Ultrathin sections (70-80 nm) were obtained by ultramicrotome (Leica Ultracut UCT, Leica, Germany) and were double stained with uranyl acetate and lead citrate and examined in transmission electron microscope (JEM 1010, Tokyo, Japan) at 60 kV. We measured areas of unmyelinated fiber, axonal diameters, G ratio, and the number of degenerative fiber using NIH Image J.

Western blot analysis
The total proteins of the sciatic nerve tissues were extracted with a Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeonggi-Do, Korea), following the manufacturer's instructions. Western blot assay was performed with specific antibodies for CaSR (Thermo

Immunofluorescence and western blot analyses in the HSCs
To evaluate the effects of cinacalcet on CsSR, CaMKKβ, phospho-Ser 428 LKB1, and phospho-Thr 172 AMPK expression, we performed immunofluorescence analysis with specific antibodies for CaSR, CaMKKβ, phospho-Ser 428 LKB1, and phospho-Thr 172 AMPK by using tyramide signal amplification fluorescence system and counterstained with DAPI. In addition, the total proteins of the HSCs were extracted with a Pro-Prep Protein Extraction Solution, following the manufacturer's instructions. After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technology, Danvers, MA), target proteins were visualized by an enhanced chemiluminescence substrate (ECL Plus, GE, Healthcare Bio-Science, Piscataway, NJ). To evaluate the anti-apoptotic effects of cinacalcet on HSCs in high-glucose medium, the number of TUNELpositive cells was counted in 10 randomly chosen fields at a magnification of ×400. We also measured the concentration of NOx to quantify NO production in cell-culture media. The total NO 3 and NO 2 were quantified using the Nitric Oxide Assay Kit (Bio Vision, Mountain View, CA).

Data analysis
SPSS version 16 (SPSS. Inc., Chicago, IL) was used to conduct the statistical analysis. Group differences were evaluated using an analysis of variance with the Bonferroni correction. Non-normally distributed data were analyzed by Mann-Whitney U test. The results are expressed as mean ± SD. A P value of <0.05 was considered statistically significant.

3− levels
The body weights of db/db mice were greater than those of db/m mice in both the cinacalcet treatment and control groups at the end of the study (p < 0.001, Table 1). No changes in body weight were noted in db/m and db/db mice following the 12-week treatment with cinacalcet. HbA1c and fasting blood sugar concentrations were significantly higher in db/db mice than those in db/m mice in both the cinacalcet treatment and control groups (p < 0.001, Table 1). Interestingly, cinacalcet treatment did not change fasting blood glucose, HbA1c, or serum iCa ++ and PO 4 3− concentrations in db/db or db/m mice.

Assessment of peripheral nerve function
Tactile response thresholds, motor nerve conduction latency, and action potential amplitude in the sciatic nerve Tactile response thresholds (Fig. 1a, 1.09 ± 0.28 vs. 0.0.53 ± 0.23 g, p < 0.001) and MNCL (Fig. 1b, 1.81 ± 0.06 vs. 1.51 ± 0.05 ms, p < 0.01) were increased in db/db cont compared with those in db/m cont mice at the end of the 12-week study. Interestingly, cinacalcet treatment significantly improved the tactile response threshold and MNCL in db/db mice compared to those in db/db control (Fig. 1a, 0.74 ± 0.18 vs. 0.36 ± 0.14 g, p < 0.01 and Fig. 1b,  1.64 ± 0.05, vs. 1.48 ± 0.06 ms, p < 0.01, respectively) to the levels similar to those in db/m mice. Consistent with the improvement in the tactile response threshold and MNCL, cinacalcet treatment increased action potential amplitude in db/db mice (Fig. 1c, 873 ± 224 vs. 1199 ± 124 μm, p < 0.01). However, there were no changes in the tactile response, sciatic motor conduction latency, and action potential amplitude in the db/m mice treated with or without cinacalcet.

Assessment of nerve pathology Expressions of nerve fibrosis and Collagen IV (Col IV)
The sciatic nerve in the db/db cont mice showed significant nerve fibrosis as reflected by increased trichromestained area when compared with that of db/m mice (Fig. 2a, b, 8.3 ± 2.1 vs. 3.4 ± 1.6%, p < 0.01) Immunohistochemical staining revealed increased expression of Col IV in the sciatic nerve of db/db cont compared to that of db/m cont mice (Fig. 2a, b, 2.4 ± 0.5 vs. 1.0 ± 0.5 folds, p < 0.01). Increased expression of Col IV and consistent increase in the extent of fibrotic area in the sciatic nerve of db/db control mice were improved to the levels similar to those of db/m mice with cinacalcet treatment (Fig. 2a, b, 3.8 ± 1.7% and 1.2 ± 0.4 folds, p < 0.01, respectively). Thus a 12-week cinacalcet treatment significantly improved fibrosis in the sciatic nerve of db/db mice.

8-OH-dG, F4/80-positive cells, and TUNEL-positive cells in the sciatic nerve
The presence of 8-OH-dG-positive area was more prominent in db/db cont mice compared with that in db/ m cont mice, reflecting increased amount of neuronal oxidative stress (Fig. 2a). However, cinacalcet treatment decreased the production of 8-OH-dG to the level comparable to that of db/m mice (Fig. 2b). We observed the number of TUNEL-positive neural cells, including SC and peripheral neuronal cells which are marked with SOX10 and β3-tubulin, respectively. Expression of TUNELpositive cells was significantly increased in db/db cont compared with that in db/m cont mice (Fig. 2c, d, 25 ± 6 vs. 13 ± 3 cells/high-power field (HPF), p < 0.01). Cinacalcet treatment reduced the number of TUNEL-positive neural cells in db/db mice (12 ± 1.7 cells/HPF). Additionally, F4/80-positive inflammatory cell infiltration was severe in db/db mice when compared with that in nondiabetic db/m mice (2.1 ± 0. comparable to those of non-diabetic mice, reflecting the recovery of autophagy process (Fig. 2c, d).

Electron microscopy
The ultrastructural features of sciatic nerves in db/db mice were characterized by severe myelin disruption with axonal shrinkage, degenerated SCs, and decreased amount of unmyelinated fibers with vacuolization when compared with those in db/m cont mice (Fig. 3a). Sciatic nerves from the db/db mice treated with cinacalcet displayed ultra-micro structures that resembled those from db/m control mice. Significant decreases in both axonal diameter and area and unmyelinated area of db/db mice were increased with cinacalcet treatment to the levels comparable to those of non-diabetic mice groups (p < 0.05 and p < 0.01, respectively; Fig. 3b). In contrast, increased G ratio in db/db mice was decreased with cinacalcet treatment, indicating optimized axonal myelination 24 with improved functional and structural indices (p < 0.01; Fig. 3b). On western blot analysis, CaSR, CaMKKβ, phospho-LKB1, phospho-AMPK, PGC-1α, and phospho-eNOS expression was significantly decreased in the sciatic nerve of db/db cont compared with that of db/m cont mice (Fig. 4a, b). These findings suggest that diabetes itself decreases the activation of CaSR-CaMKKβ and LKB1 phosphorylation, resulting in a decrease in AMPK phosphorylation, which seems to be related to the development of diabetic peripheral nerve damage. In contrast, cinacalcet treatment increased the expression of CaSR, CaMKKβ, phospho-Ser 428 LKB1, and phospho-Thr 172 AMPK, which subsequently recovered PGC-1α and eNOS expression in the sciatic nerve of db/db mice (Fig. 4a, b). For further evaluation of apoptosis and autophagy, we also measured the expression of Bcl-2/ Bax ratio, Beclin-1, and LC3-II/LC3-I ratio in the sciatic nerves. Significant decreases in the expression of Bcl-2/Bax, Beclin-1, and LC3-II/LC3-I ratio in the sciatic nerve of db/db mice increased with cinacalcet treatment to the levels comparable to those of db/m mice (Fig. 4c, d).

Effects of cinacalcet on [Ca ++ ]i in HSCs
It is well known that an increase in [Ca ++ ]i is associated with the activation of CaMKK and LKB1, which are potent activators of AMPK. Therefore, we measured the effect of cinacalcet on [Ca ++ ]i in the HSCs grown in either low-or high-glucose medium with or without cinacalcet. Interestingly, both 15 and 100 nM of cinacalcet significantly increased the peak [Ca ++ ]i levels and its area under curve (Fig. 5a, b). We further evaluated the effects of cinacalcet on AMPK activation in cultured HSCs by immunofluorescence staining and western blot analysis. The intracellular LKB-1-AMPK-PGC-1α-eNOS signaling pathway is thought to play an important role in the maintenance of normal SC function. Thus we investigated the upstream signals of AMPK; significant decreases in CaSR, CaMKKβ, phospho-Ser 428 LKB1, and phospho-Thr 172 AMPK expression were noted in HSCs grown in high-glucose medium, resulting in subsequent decreases in PGC-1α and phospho-Ser 1177 eNOS expression, which are downstream targets of AMPK (Fig. 6a, b). Consistent with the intracellular signaling changes, decreased extracellular NOx concentration and Bcl-2/Bax ratio in high-glucose medium was increased significantly with cinacalcet treatment with subsequent decrease in TUNEL-positive cells in high-glucose medium (Fig. 6b−d). Such changes were not observed either in low-glucose medium or in osmotic control (not shown), suggesting that high-glucose medium takes part in the suppression of [Ca ++ ]i-CaMKKβ-LKB-1-phospho-AMPK signaling and subsequent PGC-1α-phospho-eNOS-NOx axis in HSCs, which resulted in increased apoptotic cell deaths of HSCs. We also evaluated whether AMPK phosphorylation by cinacalcet preserves autophagy activity in SCs. The high-glucose-induced decreases in beclin-1, LC3-II/LC3-I ratio, and number of LC3 punctate in SCs were completely recovered by cinacalcet treatment to the  levels similar to those of HSCs grown in low-glucose medium (Fig. 6e). Transfection of HSCs with CaMKKβ and LKB1 siRNAs suppressed the expression of CaMKKβ and phospho-Ser 428 LKB1 by 80% and 60%, respectively, in low-glucose medium (Fig. 7a). Transfection with either CaMKKβ or LKB1 siRNA resulted in the dual suppression in the expression of CaMKKβ and phospho-LKB1 despite cinacalcet treatment. Moreover, Cinacalcet treatment did not increase the expression of phospho-Thr 172 AMPK-PGC-1α-phospho-Ser 1177 eNOS-NOx signaling in HSCs when transfected with either CaMKKβ or LKB1 siRNA (Fig. 7b, c).

Assessment of sciatic nerve function and phenotypes in 8-
week-old db/m and db/db mice To evaluate the effect of cinacalcet on the prevention and restoration of DPN, we investigated the functional and phenotypic changes in the sciatic nerves of 8-weekold db/m and db/db mice before the treatment. While tactile threshold was significantly increased in the sciatic nerve of db/db mice (p < 0.05), there were no significant differences in motor conduction latency and action potential amplitude between that of db/m and db/db mice (Fig. 8a). However, area of fibrosis, oxidative stress, and neuronal degeneration including decreased axonal diameter and area were prominent in the sciatic nerve of db/db mice with increased G ratio and decreased area for unmyelinated fibers (Fig. 8b). Furthermore, increased expression of F4/80-and TUNELpositive cells and decreased expression of LC3-positive cells were noted in the sciatic nerve of db/db mice compared with those in db/m mice (Fig. 8c) and these changes were in line with decreased expression of CaSR-AMPK-eNOS signaling pathway in the same group (Fig. 8d). LG LG +15 LG +100

Discussion
The current study provides empirical evidences that cinacalcet improved sensorimotor function and restored damaged nerve phenotypes including nerve fibrosis and inflammation, axonal degeneration, loss of unmyelinated fibers, and apoptotic neuronal cell loss in the sciatic nerve of diabetic mice. Along with these changes, cinacalcet also restored defective autophagy activity in both SCs and peripheral nerve, which characterizes early-stage diabetic neuropathy. Activation of CaMKKβ and phosphorylation of LKB1 by cinacalcet increased phosphorylation of AMPK that subsequently activated PGC-1α and phospho-Ser 1177 eNOS-NO and increased the ratio of Bcl-2/Bax, beclin-1, and LC3-II/LC3-I.
It is established that impaired [Ca ++ ]i homeostasis is implicated in the development of DPN 25,26 . Previous studies supported the notion that deranged Ca ++ homeostasis is attributable to impaired sarco/endoplasmic reticulum calcium ATPase pumps located in the endoplasmic reticulum membrane 19 . Therapy with low-dose insulin and neurotrophin-3 restored resting Ca ++ levels from intracellular stores, signifying that altered calcium homeostasis could be an early molecular marker linked to the onset of diabetic sensory neuropathy 27,28 . In the current study, cinacalcet treatment increased the expression of CaSR and [Ca ++ ]i in cytoplasm in association with subsequent increase in the expression of CaMKKβ and LKB-1 in the diabetic animals and cultured SCs that was independent of adenylate energy balance, such as AMP/ ATP and ADP/ATP ratios. More importantly, transfection with either CaMKKβ or LKB1 siRNA resulted in the dual suppression in the expression of both CaMKKβ and LKB1 and their downstream phospho-Thr 172 AMPK, PGC-1α, and phosphor-Ser 1177 eNOS signaling in HSCs cultured in high-glucose medium. The results signify the dual activation of CaMKKβ and LKB1 as a prerequisite and that the interaction between the two upstream kinases is required for further enhancement of their downstream effectors by cinacalcet treatment.
AMPK is a major downstream effector of its upstream kinases that plays a key role in cell survival and death in response to metabolic stress. The role of AMPK activation in restoring nerve function, preventing, and even reversing pathological pain associated with DPN is implicated in various studies; we previously demonstrated that fenofibrate treatment ameliorated neuronal damage in the sciatic nerve of type 2 diabetic mice by activating the PPARα-AMPK-PGC-1α-eNOS pathway 15 . Moreover, mitochondrial dysfunction in DPN is characterized by maladaptation in such metabolic signaling pathway as AMPK/sirtuin-PGC-1α axis that contributes to the diminishment of axonal regeneration capacity 29 . In line with this, diabetes-associated alterations in the peripheral nerve phenotype and concomitant development of sensorimotor dysfunction were accompanied by decreased expression of AMPK-PGC-1α-eNOS signaling and this was ameliorated by cinacalcet treatment through the upregulation of AMPK-eNOS phosphorylation in the sciatic nerve of diabetic mice.
One favorable and potential downstream mediator of AMPK signaling is NO 30 . Neuronal damages may be associated with secondary deficits in endothelial function resulting from impaired NO synthesis, release of NO upon endothelial injury by oxidative stress, and increased free radical activity. Cinacalcet's favorable effects on preventing sensorimotor dysfunction and neuronal damage may be implemented by AMPK-induced modulation of eNOS and enhancement of NOx production, which preserves peripheral nerve, especially SCs, and promotes endothelial cell survival and function.
To explore the cellular fate of peripheral nerves associated with enhanced eNOS-NOx activation, we determined the degree of autophagic activity as represented by LC3-II/LC3-I. LC3-II serves as a molecular biomarker for the assessment of autophagic activity. Impaired autophagic activity participates in the development of a variety of disease including neurodegenerative disorders probably due to the accumulation of damaged molecules (see figure on previous page) Fig. 6 Cinacalcet activates intracellular CaSR, CaMKKβ, phospho-Ser 428 LKB1, phospho-Thr 172 AMPK, PGC-1α, phospho-Ser 1177 eNOS, and NOx in HSCs grown in high-glucose medium, which prevents oxidative stress and apoptosis. a Immunofluorescence analysis was performed for CaSR, CaMKKβ, and phosphor-Ser 428 LKB1 in the HSCs with or without cinacalcet treatment (15 nM; original magnification, ×400) and the quantitative analyses of the results are shown. Scale bars represent 30 μm. (n = 6 independent experiments in each experiments) (a). b, c The effect of cinacalcet on intracellular signals and apoptosis in the human Schwann cells (HSCs) cultured in low-glucose (LG; 5 mmol/l D-glucose) or highglucose (HG; 40 mmol/l D-glucose) conditions without or with dose-dependent cinacalcet treatment (1, 5, 15 nM) were determined. CaSR, CaMKKα/β, total LKB1, phosphor-Ser 428 LKB1, total AMPK, phospho-Thr 172 AMPK, PGC-1α, phospho-Ser 1177 eNOS, NOx, BCL-2, BAX, and β-actin levels were assessed in the cultured HSCs. Representative western blot analyses and quantitative analyses of CaSR, CaMKKα/β, total LKB1, phospho-Ser 428 LKB1, total AMPK, phospho-Thr 172 AMPK, PGC-1α, and phospho-Ser 1177 eNOS are shown (b). The NOx concentrations from the supernatant of HSCs are shown (b). Representative western blot analyses and quantitative analyses of BCL-2 and BAX are shown (c). d, e. Marked increases in TUNEL-positive HSCs were observed in the HG medium compared to HG+15 group. The quantitative analyses of the results are shown (d). Representative western blot analyses and quantitative analyses of Beclin-1, LC3-1, and LC3-II are shown (e). The quantitative analyses of the results are shown (e). Marked decreases in the number of punctate in an SC were observed in the HG medium compared to HG+15 group (e). (n = 4 independent experiments in each experiments) *p < 0.05, **p < 0.01, and # p < 0.001 compared with the other groups and organelles that promotes cell death. On the other hand, excessive autophagy could also result in cell death and dysfunction by facilitating apoptosis with potential clinical significance, and therefore, the proportional contribution of autophagy 31 and apoptosis and the balance between the two counteracting functions is essential in promoting cell viability and further maintaining the functional and phenotypic integrity of peripheral nerves in diabetes. In this aspect, cinacalcet may exhibit its potential as a means to promote cell survival by enhancing autophagy and attenuating apoptosis.
In summary, this study strongly suggests a favorable effect of cinacalcet in DPN by enhancing the [Ca ++ ]i-CaMKKβ-LKB1 pathway and its downstream effectors AMPK-PGC-1α-eNOS, which may prevent the sciatic nerve injury from diabetes-induced oxidative stress through not only decreased apoptosis but also increased autophagy activity (Fig. 9). Therefore, cinacalcet-induced AMPK activation underscores the modulation of autophagy and apoptosis in the sciatic nerve and may be a promising therapeutic means to deter and prevent the progression of DPN.