Toshiaki Fukutomi

The Keap1-Nrf2 System Prevents Onset of Diabetes Mellitus

ABSTRACT Transcription factor Nrf2 (NF-E2-related factor 2) regulates a broad cytoprotective response to environmental stresses. Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein for cullin3-based ubiquitin E3 ligase and negatively regulates Nrf2. Whereas the Keap1-Nrf2 system plays important roles in oxidative stress response and metabolism, the roles Nrf2 plays in the prevention of diabetes mellitus remain elusive. Here we show that genetic activation of Nrf2 signaling by Keap1 gene hypomorphic knockdown (Keap1flox/−) markedly suppresses the onset of diabetes. When Keap1flox/− mice were crossed with diabetic db/db mice, blood glucose levels became lower through improvement of both insulin secretion and insulin resistance. Keap1flox/− also prevented high-calorie-diet-induced diabetes. Oral administration of the Nrf2 inducer CDDO-Im {oleanolic acid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole} also attenuated diabetes in db/db mice. Nrf2 induction altered antioxidant-, energy consumption-, and gluconeogenesis-related gene expression in metabolic tissues. Thus, the Keap1-Nrf2 system is a critical target for preventing the onset of diabetes mellitus.

Nrf2 Protects Pancreatic β-Cells from Oxidative and Nitrosative Stress in Diabetic Model Mice

Transcription factor Nrf2 (NF-E2–related factor 2) regulates wide-ranging cytoprotective genes in response to environmental stress. Keap1 (Kelch-like ECH–associated protein 1) is an adaptor protein for Cullin3-based ubiquitin E3 ligase and negatively regulates Nrf2. The Keap1-Nrf2 system plays important roles in the oxidative stress response and metabolism. However, the roles Nrf2 plays in prevention of pancreatic β-cell damage remain elusive. To demonstrate the roles of Nrf2 in pancreatic β-cells, we used four genetically engineered mouse models: 1) β-cell–specific Keap1-conditional knockout mice, 2) β-cell–specific Nos2 transgenic mice, 3) conventional Nrf2-heterozygous knockout mice, and 4) β-cell–specific Nrf2-conditional knockout mice. We found that Nrf2 induction suppressed the oxidative DNA-adduct formation in pancreatic islets of iNOS-Tg mice and strongly restored insulin secretion from pancreatic β-cells in the context of reactive species (RS) damage. Consistently, Nrf2 suppressed accumulation of intracellular RS in isolated islets and pancreatic β-cell lines and also decreased nitrotyrosine levels. Nrf2 induced glutathione-related genes and reduced pancreatic β-cell apoptosis mediated by nitric oxide. In contrast, Nrf2 depletion in Nrf2-heterozygous knockout and β-cell–specific Nrf2-conditional knockout mice strongly aggravated pancreatic β-cell damage. These results demonstrate that Nrf2 induction prevents RS damage in pancreatic β-cells and that the Keap1-Nrf2 system is the crucial defense pathway for the physiological and pathological protection of pancreatic β-cells.

Kinetic, thermodynamic, and structural characterizations of the association between Nrf2-DLGex degron and Keap1

ABSTRACT Transcription factor Nrf2 (NF-E2-related factor 2) coordinately regulates cytoprotective gene expression, but under unstressed conditions, Nrf2 is degraded rapidly through Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination. Nrf2 harbors two Keap1-binding motifs, DLG and ETGE. Interactions between these two motifs and Keap1 constitute a key regulatory nexus for cellular Nrf2 activity through the formation of a two-site binding hinge-and-latch mechanism. In this study, we determined the minimum Keap1-binding sequence of the DLG motif, the low-affinity latch site, and defined a new DLGex motif that covers a sequence much longer than that previously defined. We have successfully clarified the crystal structure of the Keap1-DC-DLGex complex at 1.6 Å. DLGex possesses a complicated helix structure, which interprets well the human-cancer-derived loss-of-function mutations in DLGex. In thermodynamic analyses, Keap1-DLGex binding is characterized as enthalpy and entropy driven, while Keap1-ETGE binding is characterized as purely enthalpy driven. In kinetic analyses, Keap1-DLGex binding follows a fast-association and fast-dissociation model, while Keap1-ETGE binding contains a slow-reaction step that leads to a stable conformation. These results demonstrate that the mode of DLGex binding to Keap1 is distinct from that of ETGE structurally, thermodynamically, and kinetically and support our contention that the DLGex motif serves as a converter transmitting environmental stress to Nrf2 induction as the latch site.

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

Nrf2 Improves Leptin and Insulin Resistance Provoked by Hypothalamic Oxidative Stress

The relationship between loss of hypothalamic function and onset of diabetes mellitus remains elusive. Therefore, we generated a targeted oxidative-stress murine model utilizing conditional knockout (KO) of selenocysteine-tRNA (Trsp) using rat-insulin-promoter-driven-Cre (RIP-Cre). These Trsp-KO (TrspRIPKO) mice exhibit deletion of Trsp in both hypothalamic cells and pancreatic β cells, leading to increased hypothalamic oxidative stress and severe insulin resistance. Leptin signals are suppressed, and numbers of proopiomelanocortin-positive neurons in the hypothalamus are decreased. In contrast, Trsp-KO mice (TrspIns1KO) expressing Cre specifically in pancreatic β cells, but not in the hypothalamus, do not display insulin and leptin resistance, demonstrating a critical role of the hypothalamus in the onset of diabetes mellitus. Nrf2 (NF-E2-related factor 2) regulates antioxidant gene expression. Increased Nrf2 signaling suppresses hypothalamic oxidative stress and improves insulin and leptin resistance in TrspRIPKO mice. Thus, Nrf2 harbors the potential to prevent the onset of diabetic mellitus by reducing hypothalamic oxidative damage.

Thoracoscopic esophagectomy for a patient with perforated esophageal epiphrenic diverticulum after kidney transplantation: a case report

A 58-year-old man who underwent cadaveric kidney transplantation twice presented to hospital with a perforated epiphrenic diverticulum. Computed tomography revealed epiphrenic diverticulitis and right pleural effusion. Upper gastrointestinal fibroscopy showed an epiphrenic diverticulum full of food residue. He was transferred to our hospital, where we performed percutaneous endoscopic gastrostomy under general anesthesia in the supine position before thoracoscopy. Thoracoscopic esophagectomy was performed in the semi-prone position under 6-10 mmHg artificial pneumothorax via the right thoracic cavity. We performed subtotal esophagectomy to remove sources of infection because the esophageal wall surrounding the diverticulum was too thick to close or to perform diverticulectomy. A cervical esophagostomy was constructed after the thoracic procedure. The patient was managed with continuous hemodiafiltration and administered immunosuppressants and steroids to preserve the transplanted kidney. Continuous hemodiafiltration was stopped on postoperative day (POD) 4. The patient was discharged from the intensive care unit on POD 10 and transferred to the original hospital on POD 24 for rehabilitation. The second operative stage was performed on POD 157 at our hospital. We performed gastric tube reconstruction via the ante-sternal route and anastomosed the tube to the cervical esophagus. The postoperative course was uneventful; the patient was transferred to the original hospital on POD 15 after the second operation. Minimally invasive surgery was sufficient to treat perforated epiphrenic diverticulum while preserving the transplanted kidney. We recommend completely removing the source of infection and reducing surgical invasiveness to preserve the transplanted kidney in cases of esophageal perforation following kidney transplantation.

Surgical resection and chemoradiotherapy for metachronous pulmonary metastasis of basaloid squamous cell carcinoma of the oesophagus

Highlights • Basaloid squamous cell carcinoma of the oesophagus (BSCCE) is a rare entity.• A recurrent lesion was observed after resection of the pulmonary BSCCE metastasis.• Pulmonary metastasis of BSCCE after lung metastasectomy was treated successfully with chemoradiotherapy.