Introduction and Objective: Oxidative stress and mitochondrial dysfunction play a pivotal role in the pathogenesis of diabetic neuropathy. We previously reported that hyperglycemia and glucose fluctuations induce mitochondrial dysfunction and cell death in immortalized adult mouse Schwann (IMS32) cells. Recently, the oral glucose-lowering drug imeglimin has been shown to attenuate ROS generation and improve mitochondrial function in hepatocytes. In addition, nicotinamide mononucleotide (NMN), a precursor of NAD+, has emerged as a key regulator of energy metabolism and cellular senescence. In this study, we investigated the effects of imeglimin and NMN on high glucose-induced mitochondrial dysfunction and cellular senescence in Schwann cells. Methods: IMS32 cells were cultured under either normal glucose (5.5 mM) or high glucose (25 mM). The levels of mitochondrial oxidative stress and intracellular NAD+ were measured. The protein expression levels of the longevity gene Sirt1 and the mitochondrial biogenesis-related factors PGC1α and TFAM were evaluated. Senescent cells were identified by senescence-associated β-galactosidase (SA-β-gal) staining. Results: Imeglimin and NMN suppressed high glucose-increases in mitochondrial oxidative stress. Imeglimin significantly increased intracellular NAD+ levels. Both agents restored the high glucose-induced downregulation of Sirt1, PGC1α, and TFAM expression and attenuated the increase in SA-β-gal-positive cells. Conclusion: These findings suggest that the accumulation of senescent Schwann cells may contribute to the development and progression of diabetic neuropathy. Imeglimin and NMN improved high glucose-induced mitochondrial dysfunction and suppressed cellular senescence in Schwann cells, suggesting its potential therapeutic benefits for diabetic neuropathy. Disclosure A. Kato: None. W. Nihei: None. H. Yako: None. N. Nakamura: None. K. Naruse: None. T. Himeno: None. Y. Kato: None. J. Nakamura: None. H. Kamiya: Research Support; Current; Boehringer Ingelheim International GmbH, CBC, Daiichi Sankyo, EA Pharma, Eli Lilly and Company, Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Kirin Co., Ltd., MSD, Mochida Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Parexel International, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Speaker's Bureau; Current; Abbott Japan Co., Ltd., Array BioPharma Inc., Astellas Pharma Inc., AstraZeneca, Bayer AG, Chugai Pharmaceutical Co., Ltd., EA Pharma, Eli Lilly and Company, Eisai Inc., Fukuda Denshi, GlaxoSmithKline plc., Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Kirin Co., Ltd., Novartis Pharma K.K., Novo Nordisk, Mitsubishi Tanabe Pharma Corporation, MSD, Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Sanwa Kagaku Kenkyusho, Taisho Pharmaceutical Holdings Co., Ltd., Teijin Pharma Limited, Viatris Inc. K. Sango: None. K. Kato: Speaker's Bureau; Current; Daiichi Sankyo.

ABSTRACT Aims/Introduction As a common chronic complication, diabetic neuropathy affects a substantial number of individuals with diabetes mellitus, with limited therapeutic options addressing the underlying pathogenesis. The glucose‐lowering action of imeglimin is mediated through improved insulin sensitivity in peripheral organs, including the skeletal muscle and liver, along with augmented insulin release from β‐cells. Imeglimin exerts its effects in part by modulating mitochondrial complex I, leading to the reduced production of reactive oxygen species and protection against metabolic stress. Despite these beneficial effects, the effect of imeglimin on diabetic neuropathy remains unclear. In this study, we evaluated whether imeglimin ameliorated peripheral nerve dysfunction in streptozotocin (STZ)‐induced diabetic rats. Materials and Methods The rats received imeglimin (200 mg/kg twice daily) or vehicle for 4 weeks, and the sensory nerve conduction velocity (SNCV), motor nerve conduction velocity (MNCV), sciatic nerve blood flow (SNBF), and intraepidermal nerve fiber density (IENFD) were assessed. Neurite outgrowth was examined in ND7/23 cells derived from the dorsal root ganglion. Results Imeglimin treatment significantly improved SNCV, SNBF, and IENFD without affecting blood glucose levels, indicating neuroprotective effects, independent of glycemic control. Furthermore, imeglimin enhanced neurite outgrowth in ND7/23 cells, demonstrating its direct neurotrophic effect. These findings indicate that imeglimin protects against diabetic neuropathy by enhancing nerve blood flow and promoting neurite growth independent of the systemic control of glycemia. Conclusions This study provides supporting evidence for the potential therapeutic application of imeglimin in diabetic neuropathy.

Background/Objectives: Co-culture models of neurons and Schwann cells have been used to explore the mechanisms of myelination during development, axonal regeneration after injury, and the pathogenesis of various demyelinating neuropathies. A spontaneously immortalized Fischer rat Schwann cell line 1 (IFRS1), established from the primary culture of adult Fischer344 rat peripheral nerves, can myelinate neurites in co-cultures with primary cultured dorsal root ganglion neurons and neuronal cell lines, such as nerve growth factor (NGF)-primed PC12 cells and NSC-34 motor neuron-like cells. In this study, we aimed to establish a stable co-culture system using IFRS1 cells and ND7/23 sensory neuron-like cells. Methods: ND7/23 cells were seeded at a low density (2 × 103/cm2) and maintained for 7 days in serum-containing medium supplemented with NGF (10 ng/mL) and the Rho kinase inhibitor Y27632 (5 μM) to promote neurite elongation. The cells were then treated with the anti-mitotic agent mitomycin C (1 μg/mL) for 12–16 h to suppress proliferative activity. Following this, the cells were co-cultured with IFRS1 cells (2 × 104/cm2) and maintained at 37 °C in serum-containing medium supplemented with ascorbic acid (50 μg/mL), NGF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). Results: Double-immunofluorescence staining performed on day 21 of the co-culture revealed myelin protein 22- or myelin basic protein-immunoreactive IFRS1 cells surrounding βIII tubulin-immunoreactive neurites emerging from ND7/23 cells. Myelin formation was further confirmed via Sudan Black B staining and electron microscopy. Conclusions: This co-culture system may provide a valuable tool for studying the processes of myelination in the peripheral nervous system, as well as the pathogenesis of various sensory neuropathies and potential novel therapeutic approaches for these conditions.

Aldose reductase (AR), a rate-limiting enzyme in the polyol pathway, mediates the conversion of several substrates, including glucose and galactose. In rodents, galactosemia induced by galactose feeding has been shown to develop peripheral nerve lesions resembling diabetic peripheral neuropathy. However, the mechanisms by which AR-mediated responses elicited Schwan cell lesions under galactosemic conditions remain unresolved. To investigate this, we examined the mechanism of high-galactose-induced damage mediated by AR using AR inhibitors such as ranirestat and epalrestat. The exposure of IMS32 Schwann cells under high-galactose conditions led to galactitol accumulation, the increased production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, impaired mitochondrial morphology and membrane potential, decreased glycolysis, and aberrant glycosylation. Under these experimental conditions, ranirestat inhibited intracellular galactitol in a dose-dependent manner, whereas epalrestat failed to inhibit it. Interestingly, even at low concentrations where epalrestat did not inhibit AR activity, it prevented increased ROS production, ER stress, decreased glycolysis, and aberrant RCA120-binding glycosylation; however, no effect of ranirestat on the glycosylation was observed. Epalrestat and ranirestat did not recover mitochondrial morphology. These findings suggest that ER stress is induced by aberrant glycosylation under galactosemic conditions and that epalrestat may be effective in maintaining proper glycosylation in Schwann cells in these conditions.

Diabetic peripheral neuropathy (DPN) is a chronic complication of diabetes mellitus for which effective treatments remain undeveloped. Metabolic changes and inflammation are proposed as primary mechanisms underlying DPN pathogenesis. Our previous studies demonstrate that exogenous pyruvate plays a crucial role in maintaining glycolysis-tricarboxylic acid cycle flux under high-glucose conditions and also exhibits anti-inflammatory properties. To evaluate its therapeutic potential, we assessed whether pyruvate administration could restore DPN in vivo and in vitro. We assessed casual blood glucose levels, body weight, motor and sensory nerve conduction velocities, mechanical sensitivity, and intraepidermal nerve fiber density in streptozotocin-induced diabetic C57/BL/6J mice that received drinking water with or without sodium pyruvate (10 mg/mL) from 2 to 13 weeks after diabetes induction. In addition, we evaluated neurite length in ND7/23 cells, a dorsal root ganglion neuron cell line, under high-glucose conditions. Pyruvate administration in diabetic mice alleviated mechanical sensitivity deficits and improved intraepidermal nerve fiber density. Additionally, neurite length in ND7/23 cells was inhibited under high-glucose conditions but was fully restored by supplementation with high concentrations (10 mM) of pyruvate. These findings suggest that exogenous pyruvate may be a promising therapeutic candidate for DPN.

Adipose-derived stem cells (ADSCs) can be obtained from adipose tissue, which is considered clinically dispensable. ADSCs have the ability to differentiate not only into adipocytes and osteoblasts but also into various other cell types, such as nerve cells and cardiomyocytes. However, the clinical application of ADSCs in stem cell therapy is hampered by the risk of transplant rejection and the need for facilities for their storage and transportation. In comparison, cell extracts (CEs) obtained from stem cells by freeze-thawing and lysis are less tumorigenic and immunogenic. However, there are currently no studies on the application of ADSC-derived CEs (ADSC-CEs) in peripheral nerve regeneration. Therefore, in this study, we investigated the effects of ADSC-CEs on proliferation and neurite extension in peripheral nerve cells. ADSCs were harvested from the inguinal region of mice, and ADSC-CEs were obtained following repeated freeze-thawing of ADSCs. We examined the effects of the ADSC-CEs, added to the culture medium, on glial fibrillary acidic protein (GFAP) expression and proliferation in Schwann cells. Moreover, we examined the effects of the ADSC-CEs on neurite length in DRG neurons and PC12D cells. ADSC-CEs stimulated the proliferation of Schwann cells, elevated GFAP expression in these cells, and promoted the elongation of DRG neuron and PC12D cell projections. Notably, heat treatment of the ADSC-CEs abolished these effects. Together, these findings suggest that ADSC-CEs may have therapeutic application in peripheral nerve regeneration.

Growing evidence suggests that hyperglycemia-related abnormalities in Schwann cells play a pivotal role in the development and progression of diabetic peripheral neuropathy (DPN). Several immortalized Schwann cell lines have been established in our laboratory and utilized for the study of DPN; IMS32 from normal ICR mice, 1970C3 from normal C57BL/6 mice, IWARS1 and IKARS1 from wild-type and aldose reductase-deficient C57BL/6 mice, and IFRS1 from normal Fischer 344 rats. These cell lines retain biological features of Schwann cells and display high proliferative activities that enable us to perform molecular and biochemical analyses. In addition, these cells have exhibited metabolic alterations under exposure to diabetes-associated conditions, such as hyperglycemia, dyslipidemia, glycative and oxidative stress load. Herein, recent studies with these cell lines regarding the pathogenic factors of DPN (augmentation of the polyol and other collateral glycolysis pathways, glycative and oxidative stress-induced cell injury, autophagic and proteostatic disturbances, etc.) and therapeutic strategies targeting these factors are introduced.

AIMS/INTRODUCTION:Imeglimin, a novel oral antidiabetic drug, enhances glucose-stimulated insulin secretion, improves insulin sensitivity, and reduces mitochondrial reactive oxygen species (ROS) generation. Diabetic neuropathy is driven by oxidative stress caused by hyperglycemia, with mitochondrial ROS overproduction playing a central role. Hypoglycemia also contributes to oxidative stress. This study evaluates the effects of imeglimin on Schwann cells under high- and low-glucose conditions. MATERIALS AND METHODS:We used IMS32 cells, an immortalized mouse Schwann cell line, to investigate cell survival and mitochondrial function under normal, high-, and low-glucose conditions. Assessments included mitochondrial oxidative stress, cytochrome c release, mitochondrial membrane potential, oxygen consumption rate (OCR), Complex I activity, and ATP synthesis. RESULTS:High- and low-glucose conditions caused cell death, elevated mitochondrial ROS, triggered cytochrome c release, disrupted mitochondrial membrane potential, and increased OCR and Complex I activity, while suppressing ATP synthesis. Imeglimin treatment mitigated cell death, reduced oxidative stress, restored mitochondrial membrane potential, normalized OCR and Complex I activity, and improved ATP synthesis under both glucose conditions. CONCLUSIONS:Fluctuations in glucose levels impair mitochondrial function in Schwann cells, contributing to peripheral nerve damage in diabetic neuropathy. Imeglimin demonstrated protective effects by alleviating mitochondrial dysfunction and preventing apoptosis signaling. These findings suggest the potential application of imeglimin in preventing and treating diabetic neuropathy; however, the clinical implications require further investigation.

Diabetic neuropathy (DN) is a major complication of diabetes mellitus. Chondroitin sulfate (CS) is one of the most important extracellular matrix components and is known to interact with various diffusible factors; however, its role in DN pathology has not been examined. Therefore, we generated CSGalNAc-T1 knockout (T1KO) mice, in which CS levels were reduced. We demonstrated that diabetic T1KO mice were much more resistant to DN than diabetic wild-type (WT) mice. We also found that interactions between pericytes and vascular endothelial cells were more stable in T1KO mice. Among the RNA-seq results, we focused on the transforming growth factor β signaling pathway and found that the phosphorylation of Smad2/3 was less upregulated in T1KO mice than in WT mice under hyperglycemic conditions. Taken together, a reduction in CS level attenuates DN progression, indicating that CS is an important factor in DN pathogenesis.

Increased low-density lipoprotein levels are risk factors for diabetic neuropathy. Diabetes mellitus is associated with elevated metabolic stress, leading to oxidised low-density lipoprotein formation. Therefore, it is important to investigate the mechanisms underlying the pathogenesis of diabetic neuropathy in diabetes complicated by dyslipidaemia with increased levels of oxidised low-density lipoprotein. Here, we examined the effects of hyperglycaemia and oxidised low-density lipoprotein treatment on Schwann cell death and its underlying mechanisms. Immortalised mouse Schwann cells were treated with oxidised low-density lipoprotein under normo- or hyperglycaemic conditions. We observed that oxidised low-density lipoprotein-induced cell death increased under hyperglycaemic conditions compared with normoglycaemic conditions. Moreover, hyperglycaemia and oxidised low-density lipoprotein treatment synergistically upregulated the gene and protein expression of toll-like receptor 4. Pre-treatment with TAK-242, a selective toll-like receptor 4 signalling inhibitor, attenuated hyperglycaemia- and oxidised low-density lipoprotein-induced cell death and apoptotic caspase-3 pathway. Our findings suggest that the hyperactivation of toll-like receptor 4 signalling by hyperglycaemia and elevated oxidised low-density lipoprotein levels synergistically exacerbated diabetic neuropathy; thus, it can be a potential therapeutic target for diabetic neuropathy.

To date, a considerable number of studies have shown the involvement of human gut microbiota in the development and progression of various kinds of diseases, including metabolic and neurological disorders. Fecal microbiota transplantation (FMT), the process of transplanting gut microbiota of healthy donors into intestines of patients, is considered a comprehensive therapy to improve enteric environments. FMT has been shown to suppress the progression of type 1 diabetes mellitus1 and restore insulin sensitivity in type 2 diabetes mellitus2 patients in randomized control trials (RCT). These findings indicate critical roles of gut microbiota dysbiosis in insulin deficiency and resistance. In addition, animal and preliminary human studies imply that FMT can be a potential therapy for neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease and multiple sclerosis. Diabetic peripheral neuropathy (DPN) is the most frequent and early-onset chronic complication of diabetes mellitus. Although DPN shows a variety of nerve injury patterns, its major form is distal symmetric polyneuropathy (DSPN). Ultrastructural studies have shown that the main pathology of DSPN is axonal degeneration of myelinated and unmyelinated fibers, which might characterize the disease as both metabolic and neurodegenerative disorders. As the etiology and onset mechanisms of DSPN remain largely unclear, no disease-modifying therapies against it are currently available. A recent study by Yang et al.3 shed light on gut microbiota dysbiosis as a novel pathogenic factor of DSPN; the details are described in the following paragraphs. In the first study, fecal microbiota from individuals with normal glucose levels, patients with diabetes mellitus but not DSPN and patients with DSPN (M-DSPN) were transplanted into genetically diabetic db/db mice. M-DSPN induced more severe neurological manifestations (reduced nociception and nerve conduction velocities) and histopathology (reduced intraepidermal nerve fiber density and downregulated expression of 200-kD neurofilament, myelin basic protein, and brain-derived neurotrophic factor in spinal sensory ganglia and sciatic nerves) than microbiota from individuals with normal glucose levels or patients with diabetes mellitus but not DSPN. In addition, the M-DSPN-treated group of mice showed more severe gut barrier dysfunction (reduced expression of tight junction proteins in colon tissue, and increased fluorescent dextran permeability) and systemic inflammation (increased plasma levels of lipopolysaccharide-binding protein, tumor necrosis factor-α and interleukin-6) than the other groups. Lipopolysaccharide-binding protein can bind to antigens produced by bacteria, thereby being considered a marker of bacterial antigen load in the blood. Consistent with the mouse phenotypes, genomic and metabolomic analyses with the gut microbiota resulted in significant increases in potentially pathogenic bacteria that might produce endotoxins in M-DSPN compared with microbiota from individuals with normal glucose levels and patients with diabetes mellitus but not DSPN. These findings suggest that M-DSPN exacerbates peripheral neuropathy in diabetic mice, possibly through the disruption of gut barrier integrity and the provocation of systemic inflammation through bacterial antigen load. Conversely, DSPN can be alleviated by improving enteric environments, as shown in the next clinical study. Fecal microbiota from healthy donors (FMD) or saline with plant powder (Placebo) were transplanted to patients with DSPN. The RCT for 12 weeks showed that FMD significantly alleviated DPSN, whereas Placebo showed no beneficial effects. There were no significant differences in glucose and lipid metabolism between the FMD- and Placebo-transplanted groups, whereas the severity of neuropathy evaluated by the Toronto Clinical Scoring System and visual analog scale was significantly reduced in the former, but not the latter, group. The FMD-treated group also showed diminished grades of anxiety, depression and sleep disorders, and increased grade of quality of life, as a result of respective specialized analyses. These outcomes imply that FMD-induced alleviation of neuropathic symptoms provides favorable influences on the mental states of DSPN patients. In addition to the improvement of subjective symptoms, FMD restored objective test scores for DSPN, such as sensory and motor nerve conduction velocities, and perception threshold. Furthermore, the post-RCT for 12 weeks, where the Placebo-treated patients in the RCT received FMD, resulted in a significant improvement in all the subjective and objective scores described above. These findings corroborate the efficacy of FMT toward DSPN; however, its precise action mechanisms remain to be solved. In the RCT study, biopsy specimens of colon tissue were obtained from patients before and 12 weeks after FMD or Placebo transplantation. Immunohistochemical analyses with the specimens showed the tendency of more abundant expression of tight junction proteins in FMD than Placebo. In addition, the serum levels of lipopolysaccharide-binding protein, tumor necrosis factor-α and interleukin-6 were significantly reduced in FMD, but not Placebo. These findings are in contrast to the M-DSPN effects on diabetic mice, and suggest that the beneficial effects of FMT on DSPN patients can be, at least partly, attributed to the restoration of gut barrier integrity and suppression of systemic inflammation. Metagenomic analyses on FMD and the fecal samples from the patients before and after transplantation showed that FMD treatment induced rapid and significant changes in the gut mycobiome in the patients. Among the gut microbial genomes, 21 and 33 genomes were positively and negatively correlated with the Toronto Clinical Scoring System score (the severity of neuropathic symptoms), respectively. These genomes are organized into two competing groups ("guilds"); the genomes negatively correlated with the Toronto Clinical Scoring System score were formed as a potentially beneficial guild 1, whereas those positively correlated with the score were formed as a potentially harmful guild 2. Guild 1 has a higher capacity to synthesize butylate, which might protect the mucosal barrier and maintain the host immune response. Guild 2 is involved in the antigen biosynthesis and load (Figure 1). The findings that FMD treatment increased guild 1 and decreased guild 2 in the gut mycobiome of the patients accord well with the possible action mechanisms of FMT described above. Despite these problems, this study has provided us with a novel etiology of DSPN and the therapeutic efficacy of FMT against it. Currently, no effective remedies for DPN have been established, and the only way to suppress its progression is glycemic control and improvement of lifestyle habits. Therefore, it is hoped that FMT will be available for patients as a promising treatment in the near future.

Hyperglycemia-induced oxidative stress is a major cause of the pathogenesis of diabetic neuropathy, and hyperglycemia-induced mitochondrial ROS production is considered as a significant mechanism of increased oxidative stress. On the other hand, imeglimin is the first in a new glimin class of anti-diabetic drugs targeting mitochondrial bioenergetics. In addition, imeglimin has been reported to enhance insulin secretion by increasing nicotinamide adenine dinucleotide (NAD+) levels in rat islets via the salvage pathway involving nicotinamide phosphoribosyltransferase (NAMPT). Moreover, NAD+ has been shown to modulate sirtuin and PGC1α, which in turn ameliorate mitochondrial dysfunction. We investigated the effects of imeglimin on high glucose-induced cell death and mitochondrial dysfunction in Schwann cells. In addition, the effects of imeglimin on NAD+ levels, NAMPT activity, the rate-limiting enzyme in the salvage pathway for NAD+ synthesis, and SIRT1 expression, a longevity gene, were evaluated. Immortalized adult mouse Schwann (IMS32) cells were cultured in 5.5 mM normal glucose (NG) and 25 mM high glucose (HG) medium. Compared to NG, HG decreased cell viability and increased mitochondrial oxidative stress. HG decreased mitochondrial membrane potential, increased mitochondrial oxygen consumption rate (OCR), increased activity of complex I and decreased ATP levels. Imeglimin ameliorated the reduction in cell viability and improved these mitochondrial dysfunctions. In addition, imeglimin increased NAMPT activity and NAD+ levels in IMS32 cells under HG conditions. Furthermore, imeglimin also ameliorated the reduction in SIRT1 expression by HG. These results suggest that imeglimin may prevent diabetic neuropathy by attenuating hyperglycemia-induced mitochondrial dysfunction and cell death in Schwann cells, at least in part, through the enhancement of SIRT1 and NAD+ biosynthesis. Disclosure A. Kato: None. W. Nihei: None. H. Yako: None. K. Sango: None. N. Nakamura: None. K. Naruse: None. T. Himeno: None. Y. Kato: None. J. Nakamura: None. H. Kamiya: Speaker's Bureau; Novo Nordisk, Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Boehringer-Ingelheim, Daiichi Sankyo, AstraZeneca, Ono Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Kowa Company, Ltd., Novartis Pharmaceuticals Corporation, Merck & Co., Inc., Sanwa Kagaku, Otsuka Pharmaceuticals Corporation. Research Support; Kissei Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Eli Lilly and Company, Taiho Pharmaceutical Co. Ltd., Sumitomo Dainippon Pharma Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Japan Tobacco Inc, Novo Nordisk. Speaker's Bureau; Taiho Pharmaceutical Co. Ltd., Astellas Pharma Inc., Kyowa Kirin Co., Ltd., Teijin Pharma Limited, Bayer Inc. K. Kato: None.

Pyruvate serves as a key metabolite in energy production and as an anti-oxidant. In our previous study, exogenous pyruvate starvation under high-glucose conditions induced IMS32 Schwann cell death because of the reduced glycolysis-tricarboxylic acid (TCA) cycle flux and adenosine triphosphate (ATP) production. Thus, this study focused on poly-(ADP-ribose) polymerase (PARP) to investigate the detailed molecular mechanism of cell death. Rucaparib, a PARP inhibitor, protected Schwann cells against cell death and decreased glycolysis but not against an impaired TCA cycle under high-glucose conditions in the absence of pyruvate. Under such conditions, reduced pyruvate dehydrogenase (PDH) activity and glycolytic and mitochondrial ATP production were observed but not oxidative phosphorylation or the electric transfer chain. In addition, rucaparib supplementation restored glycolytic ATP production but not PDH activity and mitochondrial ATP production. No differences in the increased activity of caspase 3/7 and the localization of apoptosis-inducing factor were found among the experimental conditions. These results indicate that Schwann cells undergo necrosis rather than apoptosis or parthanatos under the aforementioned conditions. Exogenous pyruvate plays a pivotal role in maintaining the flux in PARP-dependent glycolysis and the PARP-independent TCA cycle in Schwann cells under high-glucose conditions.