Hiroshi Takeda

Introduction: Cis-diamine-dichloro-platinum (II) (cisplatin) is a widely prescribed anticancer drug known to induce severe side effects, including skeletal muscle atrophy. As muscle atrophy negatively impacts the prognosis and survival of cancer patients, elucidation of its pathogenesis and development of preventive and treatment methods is important. Hochuekkito (HET), a Japanese Kampo medicine, has been reported to improve decreased physical activity and muscle weight in various animal models, but not in cisplatin-induced muscle atrophy. Therefore, this study aimed to clarify the characteristics of cisplatin-induced muscle atrophy and the therapeutic effects of HET, especially with emphasis in the recovery phase.Method: Eight-week-old male C57BL/6J mice were administered with cisplatin (3 mg/kg/day, intraperitoneally) from Day0 to Day3 and provided HET-containing food from 2 weeks before cisplatin administration until Day14. Muscle weight and performance were evaluated and the underlying mechanisms were investigated by using gene expression, immunohistochemistry, and microRNA (miRNA)-sequence analyses.Results: Cisplatin administration continued to reduce gastrocnemius muscle weight until Day14, even after recovery from transient decrease in food intake and body weight and was accompanied by decreased locomotor activity and grip strength, presumably due to the continuous suppression of the mRNA expression of Myogenic differentiation 1. Although HET did not suppress the activation of muscle protein degradation or increase myogenic regulatory factor expression, it restored decreased locomotor activity and gastrocnemius muscle weight by inducing an increase in the weight of the red muscle region, which contains slow-twitch fibers. Immunohistochemical analysis showed that red muscle slow-twitch fiber cross-sectional area was increased by HET treatment. The levels of miRNAs involved in muscle atrophy and aging, such as miR-29a/b and miR-34a, were increased by cisplatin; conversely, HET increased the expression of miR-1a-1 and miR-1a-2, which reportedly enhance muscle differentiation, and miR-206, which enhances slow muscle fiber differentiation and thickening.Conclusion: HET increased locomotor activity and muscle weight in cisplatin-induced muscle atrophy model mice, probably by enhancing myogenesis in slow-twitch fibers, which was related to miRNA expression changes. Thus, HET may be useful in treating cisplatin-induced muscle atrophy.

Accumulating evidence has demonstrated that both SGLT2 inhibitors (SGLT2i) and GLP-1 receptor agonists (GLP1Ra) have protective effects in patients with diabetic kidney disease. Combination therapy with SGLT2i and GLP1Ra is commonly used in patients with type 2 diabetes (T2D). We previously reported that in combination therapy of SGLT2i and GLP1Ra, the effect on the renal composite outcome did not differ according to the preceding drug. However, it remains unclear how the initiation of combination therapy is associated with the renal function depending on the preceding drug. In this post hoc analysis, we analyzed a total of 643 T2D patients (GLP1Ra-preceding group, n = 331; SGLT2i-preceding group, n = 312) and investigated the differences in annual eGFR decline. Multiple imputation and propensity score matching were performed to compare the annual eGFR decline. The reduction in annual eGFR decline in the SGLT2i-preceding group (pre: −3.5 ± 9.4 mL/min/1.73 m²/year, post: −0.4 ± 6.3 mL/min/1.73 m²/year, p < 0.001), was significantly smaller after the initiation of GLP1Ra, whereas the GLP1Ra-preceding group tended to slow the eGFR decline but not to a statistically significant extent (pre: −2.0 ± 10.9 mL/min/1.73 m²/year, post: −1.8 ± 5.4 mL/min/1.73 m²/year, p = 0.83) after the initiation of SGLT2i. After the addition of GLP1Ra to SGLT2i-treated patients, slower annual eGFR decline was observed. Our data raise the possibility that the renal benefits—especially annual eGFR decline—of combination therapy with SGLT2i and GLP1Ra may be affected by the preceding drug.

Chemotherapy-induced sarcopenia is an unfavorable prognostic factor implicated in the development of postoperative complications and reduces the quality of life of patients with cancer. Skeletal muscle loss due to cisplatin use is caused by mitochondrial dysfunction and activation of muscle-specific ubiquitin ligases Atrogin-1 and muscle RING finger 1 (MuRF1). Although animal studies suggest the involvement of p53 in age-, immobility-, and denervation-related muscle atrophy, the association between cisplatin-induced atrophy and p53 remains unknown. Herein, we investigated the effect of a p53-specific inhibitor, pifithrin-alpha (PFT-α), on cisplatin-induced atrophy in C2C12 myotubes. Cisplatin increased the protein levels of p53, phosphorylated p53, and upregulated the mRNA expression of p53 target genes PUMA and p21 in C2C12 myotubes. PFT-α ameliorated the increase in intracellular reactive oxygen species production and mitochondrial dysfunction, and also reduced the cisplatin-induced increase in the Bax/Bcl-2 ratio. Although PFT-α also reduced the cisplatin-induced increase in MuRF1 and Atrogin-1 gene expression, it did not ameliorate the decrease in myosin heavy chain mRNA and protein levels and muscle-specific actin and myoglobin protein levels. We conclude that cisplatin increases muscle degradation in C2C12 myotubes in a p53-dependent manner, but p53 has minimal involvement in the reduction of muscle protein synthesis.

Muscle atrophy is commonly observed during cisplatin chemotherapy, leading to a reduced quality of life in cancer patients. Reduced skeletal muscle mass caused by cisplatin treatment results from the activation of ubiquitin ligases–Atrogin-1 and MuRF1, but the precise mechanisms are poorly understood. In this study, we investigated the possible involvement of mitochondrial dysfunction, including reactive oxygen species (ROS) generation and ATP production, in cisplatin-induced muscle atrophy. Skeletal C2C12 myotubes were treated with cisplatin, and gene and protein expression were evaluated. Mitochondrial mass, membrane potential, and ROS levels were measured using fluorescent dyes. Mitochondrial respiratory function, ATP production rates, and glycolytic capacity were also analyzed using an extracellular flux analyzer. Metabolomic analyses were performed using gas chromatography-tandem mass spectrometry. Cisplatin treatment reduced myosin heavy chain expression by activating the ubiquitin-proteasome system. Increased ROS production was observed after cisplatin treatment, followed by significant changes in apoptosis-related gene expression and decrease in mitochondrial mass, membrane potential, respiration, and ATP production. Glycolytic capacity and TCA cycle metabolite levels were reduced with cisplatin treatment. Mitochondria-targeted antioxidant mitoquinone mesylate prevented up-regulation of Atrogin-1 gene expression and restored myosin heavy chain levels, accompanied by a decrease in ROS generation, but not mitochondrial ATP production. We concluded that cisplatin-induced myotube atrophy was associated with mitochondrial dysfunction. Reducing ROS generation, rather than promoting ATP production, could be a useful therapeutic strategy for preventing cisplatin-induced muscle atrophy.

Chronic undernutrition contributes to the increase in frailty observed among elderly adults, which is a pressing issue in the sector of health care for older people worldwide. Autophagy, an intracellular recycling system, is closely associated with age-related pathologies. Therefore, decreased autophagy in aging could be involved in the disruption of energy homeostasis that occurs during undernutrition; however, the physiological mechanisms underlying this process remain unknown. Here, we showed that 70% daily food restriction (FR) induced fatal hypoglycemia in 23–26-month-old (aged) mice, which exhibited significantly lower hepatic autophagy than 9-week-old (young) mice. The liver expressions of Bcl-2, an autophagy-negative regulator, and Beclin1–Bcl-2 binding, were increased in aged mice compared with young mice. The autophagy inducer Tat-Beclin1 D11, not the mTOR inhibitor rapamycin, decreased the plasma levels of the glucogenic amino acid and restored the blood glucose levels in aged FR mice. Decreased liver gluconeogenesis, body temperature, physical activity, amino acid metabolism, and hepatic mitochondrial dynamics were observed in the aged FR mice. These changes were restored by treatment with hochuekkito that is a herbal formula containing several autophagy-activating ingredients. Our results indicate that Bcl-2 upregulation in the liver during the aging process disturbs autophagy activation, which increases the vulnerability to undernutrition. The promotion of liver autophagy may offer clinical therapeutic benefits to frail elderly patients.

Stress is thought to be a risk factor for psychiatric disorders, such as major depression. Previously, we reported that mice exposed to repeat excessive restraint stress showed emotional abnormality. Corticosterone (CT) elevated by repeated stress have been reported to increases the ubiquitination in the brain. The present study was designed to investigate the levels of expression of ubiquitin ligase proteins in the prefrontal cortex and hippocampus of stress-maladaptive mice and to examine whether CT alters the proteins expression levels in a mouse hippocampal HT22 cells.Male ICR mice were chronically exposed to inadaptable stress, i.e. repeated restraint stress for 240 min/day for 14 days. After the final exposure to stress, brains of mice were rapidly removed and the hippocampus was dissected. HT22 cells were cultured in DMEM media supplemented with 10% FBS. Cell viability was measured by using MTT Assay. Western blot analysis revealed that no change in the level of expression of E3 ubiquitin ligase Nedd4-2 was observed in among all mice. In contrast, a significant decrease in the expression level of phosphorylated Nedd4-2 (p-Nedd4-2) was observed in the hippocampus, but not the prefrontal cortex, of stressed mice. Although CT had no effect on cell survival at concentrations of less than 3 μM, pretreatment with 3 μM CT down-regulated p-Nedd4-2 in HT22 cells. These results suggested that CT is an important effector for activation of Nedd4-2 in hippocampal neurons, and Nedd4-2 plays a pivotal role in the emotional abnormality in stress-maladaptive mice.