Improving lysosomal ferroptosis with NMN administration protects against heart failure.

Myocardial mitochondria are primary sites of myocardial energy metabolism. Mitochondrial disorders are associated with various cardiac diseases. We previously showed that mice with cardiomyocyte-specific knockout of the mitochondrial translation factor p32 developed heart failure from dilated cardiomyopathy. Mitochondrial translation defects cause not only mitochondrial dysfunction but also decreased nicotinamide adenine dinucleotide (NAD+) levels, leading to impaired lysosomal acidification and autophagy. In this study, we investigated whether nicotinamide mononucleotide (NMN) administration, which compensates for decreased NAD+ levels, improves heart failure because of mitochondrial dysfunction. NMN administration reduced damaged lysosomes and improved autophagy, thereby reducing heart failure and extending the lifespan in p32cKO mice. We found that lysosomal damage due to mitochondrial dysfunction induced ferroptosis, involving the accumulation of iron in lysosomes and lipid peroxide. The ameliorative effects of NMN supplementation were found to strongly affect lysosomal function rather than mitochondrial function, particularly lysosome-mediated ferroptosis. NMN supplementation can improve lysosomal, rather than mitochondrial, function and prevent chronic heart failure.

Feto-maternal cholesterol transport regulated by ß-Klotho-FGF15 axis is essential for fetal growth.

ß-Klotho (ß-KL) is indispensable to regulate lipid, glucose, and energy metabolism in adult animals. ß-KL is highly expressed in the yolk sac, but its role in the developmental stages has not been established. We hypothesized that ß-KL is required for metabolic regulation in the embryo and aimed to clarify the role of ß-KL during development. Here, we show that ß-KL regulates feto-maternal cholesterol transport through the yolk sac by mediating FGF 15 signaling, and also that impairment of the ß-KL-FGF15 axis causes fetal growth restriction (FGR). Embryos of ß- kl knockout (ß-kl-/-) mice were morphologically normal but exhibited FGR before placental maturation. The body weight of ß-kl-/- mice remained lower after birth. ß-KL deletion reduced cholesterol supply from the maternal blood and led to lipid shortage in the embryos. These phenotypes were similar to those of embryos lacking FGF15, indicating that ß-KL-FGF15 axis is essential for growth and lipid regulation in the embryonic stages. Our findings suggest that lipid abnormalities in early gestation provoke FGR, leading to reduced body size in later life.

Greb1 Transiently Accelerates Pancreatic ß-Cell Proliferation in Diabetic Mice Exposed to Estradiol.

Decrease of pancreatic ß cells leads to diabetes. In an inducible cAMP early suppressor (ICER-Iγ) transgenic mouse model of severe type 2 diabetes with reduced insulin production and depleted ß cells, supplementation with high concentrations of 17ß-estradiol (E2) markedly enhances ß-cell proliferation and normalizes glucose levels. The current study explored the underlying mechanisms leading to a dynamic increase of ß cells and pathologic changes in diabetic mice exposed to E2. Gene expression profiling of pancreatic islets of 6-month-old ICER-transgenic mice recovering from diabetes due to elevated E2 levels identified growth regulation by estrogen in breast cancer 1 (Greb1) as a gene significantly up-regulated during the recovery phase. To substantiate this, ß-cell-specific Greb1-deficient mice were generated, and Greb1 was shown to be essential for recovery of depleted ß cells in diabetic mice. Graft growth and glucose lowering were observed in 50 islets with increased Greb1 expression transplanted adjacent to E2 pellets beneath the kidney capsule of streptozotocin-induced diabetic mice. Greb1 expression due to a drastic increase in exogenous or endogenous E2 was transient and closely correlated with changes in E2-related and some cell cycle-related genes. These findings provide new insights into in vivo proliferation of deficient ß cells and suggest the possibility of new therapeutic approaches targeting pancreatic ß cells that could revolutionize the concept of diabetes treatment, which has been considered difficult to cure completely.

Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning.

Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

Clinical effects of wasabi extract containing 6-MSITC on myalgic encephalomyelitis/chronic fatigue syndrome: an open-label trial.

BACKGROUND: Wasabi (Eutrema japonicum) is a common pungent spice used in Japan. 6-Methylsulfinylhexyl isothiocyanate (6-MSITC) found in the rhizome of wasabi has been shown to have anti-inflammatory and antioxidant effects, as well as improve neuroinflammation and memory. Therefore, we hypothesized that these effects would be beneficial for treating myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The present study was conducted to investigate the effectiveness of wasabi extract containing 6-MSITC on ME/CFS in an open-label trial. METHODS: Fifteen patients (3 males, 12 females, 20-58 years old) were orally administered wasabi extract (9.6 mg of 6-MSITC/day) for 12 weeks. The following parameters and test results were compared pre- and post-treatment: performance status (PS), self-rating questionnaires, pressure pain threshold (PPT) on the occiput, Trail Making test-A (TMT-A), and hemodynamic patterns determined by an active standing test. RESULTS: After treatment with 6-MSITC, PS improved significantly (p = 0.001). Although the scores on the 11-item Chalder Fatigue scale (CFS-11) and numerical rating scale (NRS) of fatigue did not show significant changes, subjective symptoms improved significantly, including headache frequency (4.1 to 3.0 times/week, p = 0.001) and myalgia (4.1 to 2.4 times/week, p = 0.019), NRS brain fog scores (5.7 to 4.5, p = 0.011), difficulty finding appropriate words (4.8 to 3.7, p = 0.015), photophobia (4.8 to 3.5, p = 0.008), and the Profile of Mood Status vigor score (46.9 to 50.0, p = 0.045). The PPT of the right occiput (17.3 to 21.3 kPa, p = 0.01) and TMT-A scores (53.0 to 38.1 s, p = 0.007) also changed, suggesting reduced pain sensitivity, and improved cognitive function, respectively. Orthostatic patterns determined by a standing test did not show remarkable changes. There were no serious adverse reactions. CONCLUSION: This study suggests that 6-MSITC improves PS as well as subjective symptoms such as pain and cognitive dysfunction, and psychological vitality of patients with ME/CFS. It also improved cognitive performance and increased pain thresholds in these patients. 6-MSITC may be a promising therapeutic option especially for improving cognitive dysfunction associated with ME/CFS.

Amelioration of Murine Diabetic Nephropathy with a SGLT2 Inhibitor Is Associated with Suppressing Abnormal Expression of Hypoxia-Inducible Factors.

Diabetic nephropathy (DN), once manifested, is unlikely to completely recover. Factors that influence DN progression were explored by investigating the process of glomerulosclerosis and interstitial fibrosis and chronological changes in glucose, albuminuria, hyperfiltration, and expressions of sodium-glucose cotransporter 2 (SGLT2) and hypoxia-inducible factors (HIFs) up to 50 weeks in inducible cAMP early repressor transgenic mice, a model of severe DN. Long-term intervention with the SGLT2 inhibitor canagliflozin or islet transplantation or heminephrectomy was used. Inducible cAMP early repressor transgenic mice exhibited progressive diabetic glomerulosclerosis and mild interstitial fibrosis, and expressed extensive HIF-1α and HIF-2α in glomerulus and tubules, with sustained hyperfiltration up to 50 weeks. Canagliflozin ameliorated glomerulosclerosis/interstitial fibrosis gradually and reduced HIF overexpression. Islet-transplanted mice exhibited no amelioration. None of the heminephrectomized diabetic mice survived the hyperfiltration overload, but all of the canagliflozin-treated mice survived with re-expressions of HIF-1α and HIF-2α. These results suggest that persistent glomerular hyperfiltration might initiate glomerular injury, and persistent overexpression of HIFs could promote the development of glomerulosclerosis and interstitial fibrosis. Canagliflozin attenuated both changes. Oxidative stress or hypoxia was undetectable in this model. The abnormal expression of HIF-1α and HIF-2α may be a potential therapeutic target for preventing glomerulosclerosis and interstitial fibrosis.

NRF2 pathway activation attenuates ageing-related renal phenotypes due to α-klotho deficiency.

Oxidative stress is one of the major causes of the age-related functional decline in cells and tissues. The KEAP1-NRF2 system plays a central role in the regulation of redox balance, and NRF2 activation exerts antiageing effects by controlling oxidative stress in aged tissues. α-Klotho was identified as an ageing suppressor protein based on the premature ageing phenotypes of its mutant mice, and its expression is known to gradually decrease during ageing. Because α-klotho has been shown to possess antioxidant function, ageing-related phenotypes of α-klotho mutant mice seem to be attributable to increased oxidative stress at least in part. To examine whether NRF2 activation antagonizes ageing-related phenotypes caused by α-klotho deficiency, we crossed α-klotho-deficient (Kl-/-) mice with a Keap1-knockdown background, in which the NRF2 pathway is constitutively activated in the whole body. NRF2 pathway activation in Kl-/- mice extended the lifespan and dramatically improved ageing-related renal phenotypes. With elevated expression of antioxidant genes accompanied by an oxidative stress decrease, the antioxidant effects of NRF2 seem to make a major contribution to the attenuation of ageing-related renal phenotypes of Kl-/- mice. Thus, NRF2 is expected to exert an antiageing function by partly compensating for the functional decline of α-Klotho during physiological ageing.

Alternative Functions of Cell Cycle-Related and DNA Repair Proteins in Post-mitotic Neurons.

Proper regulation of neuronal morphological changes is essential for neuronal migration, maturation, synapse formation, and high-order function. Many cytoplasmic proteins involved in the regulation of neuronal microtubules and the actin cytoskeleton have been identified. In addition, some nuclear proteins have alternative functions in neurons. While cell cycle-related proteins basically control the progression of the cell cycle in the nucleus, some of them have an extra-cell cycle-regulatory function (EXCERF), such as regulating cytoskeletal organization, after exit from the cell cycle. Our expression analyses showed that not only cell cycle regulators, including cyclin A1, cyclin D2, Cdk4/6, p21cip1, p27kip1, Ink4 family, and RAD21, but also DNA repair proteins, including BRCA2, p53, ATM, ATR, RAD17, MRE11, RAD9, and Hus1, were expressed after neurogenesis, suggesting that these proteins have alternative functions in post-mitotic neurons. In this perspective paper, we discuss the alternative functions of the nuclear proteins in neuronal development, focusing on possible cytoplasmic roles.

Simple method for large-scale production of macrophage activating factor GcMAF.

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing.

Effect of oral administration of nicotinamide mononucleotide on clinical parameters and nicotinamide metabolite levels in healthy Japanese men.

Recent studies have revealed that decline in cellular nicotinamide adenine dinucleotide (NAD+) levels causes aging-related disorders and therapeutic approaches increasing cellular NAD+ prevent these disorders in animal models. The administration of nicotinamide mononucleotide (NMN) has been shown to mitigate aging-related dysfunctions. However, the safety of NMN in humans have remained unclear. We, therefore, conducted a clinical trial to investigate the safety of single NMN administration in 10 healthy men. A single-arm non-randomized intervention was conducted by single oral administration of 100, 250, and 500 mg NMN. Clinical findings and parameters, and the pharmacokinetics of NMN metabolites were investigated for 5 h after each intervention. Ophthalmic examination and sleep quality assessment were also conducted before and after the intervention. The single oral administrations of NMN did not cause any significant clinical symptoms or changes in heart rate, blood pressure, oxygen saturation, and body temperature. Laboratory analysis results did not show significant changes, except for increases in serum bilirubin levels and decreases in serum creatinine, chloride, and blood glucose levels within the normal ranges, independent of the dose of NMN. Results of ophthalmic examination and sleep quality score showed no differences before and after the intervention. Plasma concentrations of N-methyl-2-pyridone-5-carboxamide and N-methyl-4-pyridone-5-carboxamide were significantly increased dose-dependently by NMN administration. The single oral administration of NMN was safe and effectively metabolized in healthy men without causing any significant deleterious effects. Thus, the oral administration of NMN was found to be feasible, implicating a potential therapeutic strategy to mitigate aging-related disorders in humans.

Growth Arrest Triggers Extra-Cell Cycle Regulatory Function in Neurons: Possible Involvement of p27kip1 in Membrane Trafficking as Well as Cytoskeletal Regulation.

Cell cycle regulation is essential for the development of multicellular organisms, but many cells in adulthood, including neurons, exit from cell cycle. Although cell cycle-related proteins are suppressed after cell cycle exit in general, recent studies have revealed that growth arrest triggers extra-cell cycle regulatory function (EXCERF) in some cell cycle proteins, such as p27(kip1), p57(kip2), anaphase-promoting complex/cyclosome (APC/C), and cyclin E. While p27 is known to control G1 length and cell cycle exit via inhibition of cyclin-dependent kinase (CDK) activities, p27 acquires additional cytoplasmic functions in growth-arrested neurons. Here, we introduce the EXCERFs of p27 in post-mitotic neurons, mainly focusing on its actin and microtubule regulatory functions. We also show that a small amount of p27 is associated with the Golgi apparatus positive for Rab6, p115, and GM130, but not endosomes positive for Rab5, Rab7, Rab8, Rab11, SNX6, or LAMTOR1. p27 is also colocalized with Dcx, a microtubule-associated protein. Based on these results, we discuss here the possible role of p27 in membrane trafficking and microtubule-dependent transport in post-mitotic cortical neurons. Collectively, we propose that growth arrest leads to two different fates in cell cycle proteins; either suppressing their expression or activating their EXCERFs. The latter group of proteins, including p27, play various roles in neuronal migration, morphological changes and axonal transport, whereas the re-activation of the former group of proteins in post-mitotic neurons primes for cell death.

Alzheimer Aß Assemblies Accumulate in Excitatory Neurons upon Proteasome Inhibition and Kill Nearby NAKα3 Neurons by Secretion.

We identified ∼30-mer amyloid-ß protein (Aß) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aß levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.

Caveolin-1 Promotes Early Neuronal Maturation via Caveolae-Independent Trafficking of N-Cadherin and L1.

Axon specification is morphologically reproducible in vitro, whereas dendrite formation differs in vitro and in vivo. Cortical neurons initially develop immature neurites, but in vivo these are eliminated concurrently with the formation of a leading process, the future dendrite. However, the molecular mechanisms underlying these neuronal maturation events remain unclear. Here we show that caveolin-1, a major component of caveolae that are never observed in neurons, regulates in vivo-specific steps of neuronal maturation. Caveolin-1 is predominantly expressed in immature cortical neurons and regulates clathrin-independent endocytosis. In vivo knockdown of caveolin-1 disturbs immature neurite pruning, leading process elongation, and subsequent neuronal migration. Importantly, N-cadherin and L1, which are required for immature neurite formation, undergo caveolin-1-mediated endocytosis to eliminate immature neurites. Collectively, our findings indicate that caveolin-1 regulates N-cadherin and L1 trafficking independent of caveolae, which contributes to spatiotemporally restricted cellular events; immature neurite pruning and leading process elongation during early neuronal maturation.

Forebrain Ptf1a Is Required for Sexual Differentiation of the Brain.

The mammalian brain undergoes sexual differentiation by gonadal hormones during the perinatal critical period. However, the machinery at earlier stages has not been well studied. We found that Ptf1a is expressed in certain neuroepithelial cells and immature neurons around the third ventricle that give rise to various neurons in several hypothalamic nuclei. We show that conditional Ptf1a-deficient mice (Ptf1a cKO) exhibit abnormalities in sex-biased behaviors and reproductive organs in both sexes. Gonadal hormone administration to gonadectomized animals revealed that the abnormal behavior is caused by disorganized sexual development of the knockout brain. Accordingly, expression of sex-biased genes was severely altered in the cKO hypothalamus. In particular, Kiss1, important for sexual differentiation of the brain, was drastically reduced in the cKO hypothalamus, which may contribute to the observed phenotypes in the Ptf1a cKO. These findings suggest that forebrain Ptf1a is one of the earliest regulators for sexual differentiation of the brain.

Diagnostic and clinical significance of the titin fragment in urine of Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle wasting disease of childhood. Titin in sarcomere is digested by calcium dependent protease. To explore muscle damage in DMD, the urinary concentrations of the N-terminal fragment of titin were determined using a newly developed enzyme linked immune sorbent assay kit. The urinary titin concentrations were normalized to creatinine (Cr). A total of 145 urine samples were obtained at a single Japanese hospital from 113 DMD patients aged 3-29years. Normalized urinary titin concentration was 965.8±1011.9 (Mean±SD) pmol/mg Cr in patients with DMD. This was nearly 700-fold higher than healthy children (1.4±0.8pmol/mg Cr). The concentration was significantly higher in DMD than in BMD patients who had significantly higher urinary titin than normal. Urinary titin in DMD patients tended to decrease with age. The median concentration of urinary titin in the youngest (aged 3-7years) and oldest (aged ≥16years) groups was 1468.3 and 411.3pmol/mg Cr, respectively, with significant difference. Urinary concentration of titin correlated significantly with serum creatine kinase concentration, the best-known biomarker of DMD. The N-terminal fragment of titin in urine has potential as a diagnostic and clinical biomarker for DMD.

Mouse macrophages show different requirements for phosphatidylserine receptor Tim4 in efferocytosis.

Protein S (ProS) and growth arrest-specific 6 (Gas6) bind to phosphatidylserine (PtdSer) and induce efferocytosis upon binding TAM-family receptors (Tyro3, Axl, and Mer). Here, we produced mouse ProS, Gas6, and TAM-receptor extracellular region fused to IgG fragment crystallizable region in HEK293T cells. ProS and Gas6 bound Ca2+ dependently to PtdSer (Kd 20-40 nM), Mer, and Tyro3 (Kd 15-50 nM). Gas6 bound Axl strongly (Kd < 1.0 nM), but ProS did not bind Axl. Using NIH 3T3-based cell lines expressing a single TAM receptor, we showed that TAM-mediated efferocytosis was determined by the receptor-binding ability of ProS and Gas6. Tim4 is a membrane protein that strongly binds PtdSer. Tim4 alone did not support efferocytosis, but enhanced TAM-dependent efferocytosis. Resident peritoneal macrophages, Kupffer cells, and CD169+ skin macrophages required Tim4 for TAM-stimulated efferocytosis, whereas efferocytosis by thioglycollate-elicited peritoneal macrophages or primary cultured microglia was TAM dependent, but not Tim4 dependent. These results indicate that TAM and Tim4 collaborate for efficient efferocytosis in certain macrophage populations.

Morphological and Molecular Basis of Cytoplasmic Dilation and Swelling in Cortical Migrating Neurons.

During corticogenesis, neuronal migration is an essential step for formation of a functional brain, and abnormal migration is known to cause various neurological disorders. Neuronal migration is not just a simple movement of the cell body, but a consequence of various morphological changes and coordinated subcellular events. Recent advances in in vivo and ex vivo cell biological approaches, such as in utero gene transfer, slice culture and ex vivo chemical inhibitor techniques, have revealed details of the morphological and molecular aspects of neuronal migration. Migrating neurons have been found to have a unique structure, dilation or swelling, at the proximal region of the leading process; this structure is not found in other migrating cell types. The formation of this structure is followed by nuclear deformation and forward movement, and coordination of this three-step sequential morphological change (the dilation/swelling formation, nuclear elongation and nuclear movement) is essential for proper neuronal migration and the construction of a functional brain structure. In this review, we will introduce the morphological features of this unique structure in migrating neurons and summarize what is known about the molecules regulating the dilation/swelling formation and nuclear deformation and movement.

Fgf21 regulates T-cell development in the neonatal and juvenile thymus.

We have previously shown that Fibroblast growth factor 21 (Fgf21) is expressed in the thymus as well as in the liver. In line with this expression profile, Fgf21 was recently reported to protect against ageing-related thymic senescence by improving the function of thymic epithelial cells (TECs). However, the function of Fgf21 in the juvenile thymus remained to be elucidated. We investigated the physiological roles of Fgf21 in the juvenile thymus and found that young Fgf21 knockout mice, but not ß-Klotho knockout mice nor adult Fgf21 knockout mice, showed a significant reduction in the percentage of single-positive CD4+ and CD8+ thymocytes without obvious alteration in TECs. Furthermore, treatment with recombinant FGF21 protein rescued the impairment in fetal thymus organ culture (FTOC) of Fgf21 knockout mice. Annexin V staining revealed FGF21 protein enhanced apoptosis of immature thymocytes undergoing selection process in FTOC, suggesting that FGF21 may facilitate the selection of developing T cells. Endocrine Fgf21 from the liver induced by metabolic stimulation did not affect juvenile thymocyte development. Our data suggest that Fgf21 acts as one of intrathymic cytokines in the neonatal and juvenile thymus, involving thymocyte development in a ß-Klotho-independent manner.

Establishment of a highly sensitive sandwich ELISA for the N-terminal fragment of titin in urine.

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes.

Effects of 17ß-Estradiol and Androgen on Glucose Metabolism in Skeletal Muscle.

Diabetes develops predominantly in males in experimental models, and extensive evidence suggests that 17ß-estradiol (E2) modulates progression of diabetes in humans. We previously developed a severely diabetic transgenic (Tg) mouse model by ß-cell-specific overexpression of inducible cAMP early repressor (ICER) and found that male ICER-Tg mice exhibit sustained severe hyperglycemia, but female ICER-Tg mice gradually became normoglycemic with aging. This implies that differences in circulating androgen and E2 levels might influence skeletal muscle glucose uptake and glycemic status. Here we examined whether a decrease of androgen or E2 excess can improve muscle glucose uptake in hyperglycemic male ICER-Tg mice and, conversely, whether a decrease of E2 or androgen excess can elevate blood glucose levels and impair muscle glucose uptake in normoglycemic female ICER-Tg mice. We treated hyperglycemic male ICER-Tg mice with orchiectomy (ORX) or ORX+E2 pellet implantation and normoglycemic female ICER-Tg mice with ovariectomy (OVX) or OVX+5α-DHT pellet implantation to alter the androgen to E2 ratio. ORX+E2 treatment of male ICER-Tg mice caused a rapid drop in blood glucose via both a dramatic increase of ß-cells and significantly improved muscle glucose uptake due to the induction of glucose transporter type 4 (GLUT4) expression and translocation of GLUT4 to the cell membrane. In contrast, OVX+5α-DHT-treated female ICER-Tg mice showed an elevation of blood glucose without any decrease of ß-cells; instead, they showed decreased muscle glucose uptake due to decreased activation of serine/threonine-specific protein kinase AKT and GLUT4 expression. These findings suggest that androgen (5α-DHT) promotes insulin resistance in females, whereas E2 improves insulin sensitivity in severely diabetic male mice.