Yoko Yagishita

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.

The Keap1-Nrf2 system and diabetes mellitus.

Nrf2 (NF-E2-related factor 2) plays a key role in the protection of vertebrates against environmental stress by contributing to the inducible expression of detoxification and antioxidant enzymes. Keap1 (Kelch-like ECH-associated protein 1) is a sensor for oxidative and electrophilic stresses. Keap1 also acts as an E3 ubiquitin ligase substrate-recognition subunit that specifically targets Nrf2. Keap1 causes Nrf2 to be degraded through the ubiquitin-proteasome pathway and thus ensures that Nrf2 is constitutively suppressed under unstressed conditions. Upon exposure to oxidative or electrophilic stress, Keap1 loses its ability to ubiquitinate Nrf2. Many lines of evidence have recently clarified that the Keap1-Nrf2 system also plays critical roles in the maintenance of cellular homeostasis. One of the most salient examples is the contribution of Keap1-Nrf2 to metabolic and energy-balance regulation. In particular, how the Keap1-Nrf2 system protects the body against diabetes mellitus and how perturbations in this system provoke the disease condition are now under intense investigation. This review will summarize the recent progress made in this area.

Nrf2 induces fibroblast growth factor 21 in diabetic mice

Transcription factor Nrf2 (nuclear factor E2‐related factor 2) is a master regulator of cellular defense system against oxidative and electrophilic stresses and is negatively regulated by an adaptor protein Keap1 (Kelch‐like ECH‐associated protein 1). Nrf2 also plays a pivotal role in metabolic homeostasis, such as lipid metabolism and energy expenditure as well as redox homeostasis. FGF21 (fibroblast growth factor 21) is known as a key mediator of glucose and lipid metabolism. Here, we found that Nrf2 is involved in FGF21 regulation in diabetic model mice. Nrf2 induction by genetic knockdown of Keap1 increased plasma FGF21 level and hepatic Fgf21 expression in diabetic db/db mice and high‐calorie‐diet‐induced obesity model mice. Administration of CDDO‐Im (oleanolic triterpenoid 1‐[2‐cyano‐3,12‐dioxooleane‐1, 9(11)‐dien‐28‐oyl] imidazole), a potent Nrf2 inducer, up‐regulated plasma FGF21 level and hepatic Fgf21 expression in db/db mice, whereas CDDO‐Im did not induce FGF21 in db/db mice with Nrf2 knockout background. Furthermore, in Keap1‐knockdown db/db mice, Nrf2 enhanced expression of glucose‐ and lipid‐metabolism‐related genes in adipose tissues, which improved plasma lipid profiles. These results show that Nrf2 positively regulates FGF21 expression in diabetic mice. We propose that FGF21 is a potential efficacy biomarker that mediates metabolic regulation by the Keap1–Nrf2 system.

Lymphatic mapping of mice with systemic lymphoproliferative disorder: Usefulness as an inter-lymph node metastasis model of cancer

Preclinical models of lymph node (LN) metastasis are fundamental to the study and design of new techniques for the diagnosis and treatment of LN metastasis. However, the identification of LNs and lymphatic vessels (LVs) in mice is challenging with conventional imaging modalities, since the LN diameter in normal mice is 1-2 mm. Here, we describe MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) inbred mice, which develop systemic swelling of LNs up to 10 mm in diameter, allowing investigation of the topography of LNs and LVs. Using a gross anatomy dissection approach, we identified 22 different LNs situated in the head and neck, limbs, thoracic and abdominal regions. Furthermore, four peripheral inter-LN vessels were found: from the subiliac LN (SiLN) to the proper axillary LN (PALN); from the parotid LN to the caudal deep cervical LN; and from the popliteal LN to both the sciatic LN and the SiLN. Metastasis to the PALN via LVs was induced by inoculating FM3A/Luc mouse mammary carcinoma cells into the SiLN. Our results demonstrate that the MXH10/Mo/lpr mouse strain is an excellent model in which to investigate lymphatic drainage and inter-LN metastasis of cancer. This paper unveils the anatomy of murine lymphatics to give new insights into the investigation of inter-LN metastasis of cancer, especially the mechanisms involved in the trafficking of cancer cells through inter-LN vessels. The results provide data that may prove very useful in the quest to develop better lymph drainage-based drug delivery systems.

Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism

ABSTRACT Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo. Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver.

Volumetric and Angiogenic Evaluation of Antitumor Effects with Acoustic Liposome and High-Frequency Ultrasound

Acoustic liposomes (AL) have their inherent echogenicity and can add functionality in serving as drug carriers with tissue specificity. Nonuniform vascular structures and vascular branches/bends are evaluated by imaging the intravascular movement locus of ALs with high-frequency ultrasound (HF-US) imaging. However, the evaluation of antitumor effects on angiogenesis by ALs and HF-US imaging has not been reported. Here, we show that the combination of ALs and an HF-US imaging system is capable of noninvasively evaluating antitumor volumetric and angiogenic effects in preclinical mouse models of various cancers. In this study, the antitumor effects of cisplatin on tumor growth and angiogenesis in mice bearing two different types of tumor cells were assessed. By tracking each AL flowing in the vessel and transferring the images to personal computers, microvessel structures were mapped and reconstructed using the color difference based on SD method. The antitumor effects were confirmed with an in vivo bioluminescence imaging system and immunohistochemical analysis. Our results show that cisplatin inhibits tumor growth by decreasing intratumoral vessel area but does not affect the angiogenesis ratio in the tumor. The vascular occupancy in the outer region of the tumor was larger than that in the inner region; however, both occupancies were similar to those of the control tumor. We propose that this method of mapping microvessels with ALs and an HF-US system can serve as a new molecular imaging method for the assessment of angiogenesis and can be applied to evaluate the antitumor effects by various therapeutic agents.

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.

The novel Nrf2 inducer TFM-735 ameliorates experimental autoimmune encephalomyelitis in mice

ABSTRACT The transcription factor NF‐E2‐related factor 2 (Nrf2) is a key regulator of cellular defense mechanisms against oxidative stress. Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system, is characterized by progressive demyelination and neurodegeneration induced by inflammation and oxidative stress. The induction of Nrf2 signaling has been shown to inhibit disease development and progression in the experimental autoimmune encephalomyelitis (EAE) model of MS in mice. In the present study, we performed a high‐throughput screening assay using a chimeric construct of the N‐terminal portion of Nrf2 fused to LacZ. Using this approach, we identified the novel Nrf2 inducer TFM‐735. Using human primary cell profiling systems, we found that TFM‐735 inhibited T cell proliferation and exerted immuno‐modulatory effects by inhibiting the production of IL‐6 and IL‐17. TFM‐735 also inhibited IL‐17 secretion from human peripheral blood mononuclear cells stimulated with anti‐CD3 and anti‐CD28. In EAE mice treated with TFM‐735, the expression of the Nrf2 target gene Nqo1 increased in the brain and spleen, disease severity was ameliorated, and plasma IL‐17 levels decreased. Furthermore, TFM‐735 inhibited luciferase activity in Wim‐6 transgenic EAE mice expressing the human interleukin 6‐luciferase (hIL6‐BAC‐Luc) reporter. Therefore, these findings indicate that TFM‐735 is a potent Nrf2 inducer that inhibits inflammatory cytokine production and disease progression in mice with EAE and that TFM‐735 is a promising therapeutic agent for MS.

Short-term effect of single NaF-mouthrinse on glucose-induced pH fall in dental plaque

This study aimed to evaluate the short-term effect of a single fluoride-mouthrinse containing different concentrations of sodium fluoride (NaF) on glucose-induced pH fall and fluoride retention within dental plaque. The NaF-mouthrinse inhibited decreases in plaque-pH in short-term, probably due to fluoride retention within plaque. Thus, it is expected that plaque acid production by bacterial sugar metabolism may be inhibited by NaF-mouthrinse in vivo.