[Subacute myopathy in a patient with mild Cushing disease manifested by accompanying Kleinfelter syndrome].

A 69-year-old man was admitted because of subacute development of lower limb weakness from one month ago. He showed central obesity, gynecomastia, dorsal fat pad ("buffalo hump"), and proximal muscle weakness in the lower extremities (manual muscle test 4). Needle EMG, muscle MRI and labolatry screening including CPK were negative for neuromuscular diseases, except for the hypogenitalism accidentally detected in MRI. Although blood corticol was in normal range, the levels of serum ACTH and 24-hour urinary free cortisol excretion were high, and the dexamethasone suppression tests were positive. Brain MRI showed a small pituitary mass with gadolinium enhancement, and ACTH measurement from petrosal sinus sampling after CRH stimulation lead to the diagnosis of definite Cushing disease. Moreover, he also showed low testosterone and elevated LH and FSH. Chromosome banding revealed 47 XXY in 22 in 30 cells, leading to the diagnosis of mosaic Klinefelter syndrome. The supplementation with testosterone was partially effective for his weakness. The surgical resection of pituitary microadenoma resulted in the full recovery. Either Klinefelter syndrome or mild Cushing disease alone was insufficient as a cause of the muscle weakness in this patient. It is plausible that the mild elevation of cortisol accompanied by the lack of tesstelone may underlie the weakness, probably linked to impaired balance between muscle anabolism and catabolism.

Charcot-Marie-Tooth disease type 2A with an autosomal-recessive inheritance: the first report of an adult-onset disease.

Axonal Charcot-Marie-Tooth disease (CMT) is most frequently caused by mutations in the MFN2 gene (CMT2A) that can lead to various clinical phenotypes. The age at disease onset varies, but most cases occur before adolescence. We report two Japanese sisters who presented with middle-age-onset peripheral neuropathy with distinct clinical features. In the affected sisters, a homozygous missense mutation, c.1894C>T, p.R632W, corresponding to the transmembrane domain of MFN2 was identified; this mutation was heterozygous in another non-affected sibling, demonstrating co-segregation of the genotype and phenotype. The patients developed adult-onset slowly progressive muscle weakness that was predominant in the calf muscles and sensory disturbance. Magnetic resonance imaging revealed diffuse atrophy of the spinal cord, especially in the thoracic segment, and mild atrophy of the parietal lobe and the cerebellum in both patients. Electron microscopy of the sural nerve revealed clusters of round and swollen mitochondria. This is the first case report of adult-onset CMT2A with an autosomal-recessive inheritance pattern. The phenotype caused by the MFN2 mutation in these cases is very mild, considering that the mutation causes middle-aged-onset Charcot-Marie-Tooth even in the homozygous state. The mechanism of MFN2 mutation-induced toxicity is an interesting theme that awaits further investigations.

Clinical Phenotype and Segregation of Mitochondrial 3243A>G Mutation in 2 Pairs of Monozygotic Twins.

IMPORTANCE: The regulatory factors explaining the wide spectrum of clinical phenotypes for mitochondrial 3243A>G mutation are not known. Crosstalk between nuclear genes and mitochondrial DNA might be one factor. OBSERVATIONS: In this case series, we compared 2 pairs of male twins with the mitochondrial 3243 A>G mutation and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome with a female control patient. One pair of monozygotic twins presented with diabetes and deafness in their 30s, stroke-like episodes in their 40s, and cardiac events and death in their 50s. Another pair of twins presented with deafness and stroke-like episodes in their 20s. The degree of heteroplasmy of 3243A>G mutation in the various tissues and organs was similar in the first pair of twins compared with the control patient. CONCLUSIONS AND RELEVANCE: The clinical phenotype and segregation of mitochondrial 3243A>G mutation was similar in monozygotic twins. The onset age and distribution of the symptoms might be regulated by nuclear genes. Our findings might help to predict the clinical course of the surviving twins and afford an opportunity for therapy before the onset of mitochondrial disease, especially for monozygotic twins caused by nuclear transfer with a small amount of nuclear-donor mitochondrial DNA.

Urinary Potassium Excretion and Renal and Cardiovascular Complications in Patients with Type 2 Diabetes and Normal Renal Function.

BACKGROUND AND OBJECTIVES: We investigated the association of urinary potassium and sodium excretion with the incidence of renal failure and cardiovascular disease in patients with type 2 diabetes. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 623 Japanese type 2 diabetic patients with eGFR≥60 ml/min per 1.73 m(2) were enrolled in this observational follow-up study between 1996 and 2003 and followed-up until 2013. At baseline, a 24-hour urine sample was collected to estimate urinary potassium and sodium excretion. The primary end point was renal and cardiovascular events (RRT, myocardial infarction, angina pectoris, stroke, and peripheral vascular disease). The secondary renal end points were the incidence of a 50% decline in eGFR, progression to CKD stage 4 (eGFR<30 ml/min per 1.73 m(2)), and the annual decline rate in eGFR. RESULTS: During the 11-year median follow-up period, 134 primary end points occurred. Higher urinary potassium excretion was associated with lower risk of the primary end point, whereas urinary sodium excretion was not. The adjusted hazard ratios for the primary end point in Cox proportional hazards analysis were 0.56 (95% confidence interval [95% CI], 0.33 to 0.95) in the third quartile of urinary potassium excretion (2.33-2.90 g/d) and 0.33 (95% CI, 0.18 to 0.62) in the fourth quartile (>2.90 g/d) compared with the lowest quartile (<1.72 g/d). Similar associations were observed for the secondary renal end points. The annual decline rate in eGFR in the fourth quartile of urinary potassium excretion (-1.3 ml/min per 1.73 m(2)/y; 95% CI, -1.5 to -1.0) was significantly slower than those in the first quartile (-2.2; 95% CI, -2.4 to -1.8). CONCLUSIONS: Higher urinary potassium excretion was associated with the slower decline of renal function and the lower incidence of cardiovascular complications in type 2 diabetic patients with normal renal function. Interventional trials are necessary to determine whether increasing dietary potassium is beneficial.

Predictive properties of plasma amino acid profile for cardiovascular disease in patients with type 2 diabetes.

Prevention of cardiovascular disease (CVD) is an important therapeutic object of diabetes care. This study assessed whether an index based on plasma free amino acid (PFAA) profiles could predict the onset of CVD in diabetic patients. The baseline concentrations of 31 PFAAs were measured with high-performance liquid chromatography-electrospray ionization-mass spectrometry in 385 Japanese patients with type 2 diabetes registered in 2001 for our prospective observational follow-up study. During 10 years of follow-up, 63 patients developed cardiovascular composite endpoints (myocardial infarction, angina pectoris, worsening of heart failure and stroke). Using the PFAA profiles and clinical information, an index (CVD-AI) consisting of six amino acids to predict the onset of any endpoints was retrospectively constructed. CVD-AI levels were significantly higher in patients who did than did not develop CVD. The area under the receiver-operator characteristic curve of CVD-AI (0.72 [95% confidence interval (CI): 0.64-0.79]) showed equal or slightly better discriminatory capacity than urinary albumin excretion rate (0.69 [95% CI: 0.62-0.77]) on predicting endpoints. A multivariate Cox proportional hazards regression analysis showed that the high level of CVD-AI was identified as an independent risk factor for CVD (adjusted hazard ratio: 2.86 [95% CI: 1.57-5.19]). This predictive effect of CVD-AI was observed even in patients with normoalbuminuria, as well as those with albuminuria. In conclusion, these results suggest that CVD-AI based on PFAA profiles is useful for identifying diabetic patients at risk for CVD regardless of the degree of albuminuria, or for improving the discriminative capability by combining it with albuminuria.

Stem cell factor-activated bone marrow ameliorates amyotrophic lateral sclerosis by promoting protective microglial migration.

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with motor neuron death. Several experimental treatments, including cell therapy using hematopoietic or neuronal stem cells, have been tested in ALS animal models, but therapeutic benefits have been modest. Here we used a new therapeutic strategy, bone marrow transplantation (BMT) with stem cell factor (SCF)- or FMS-like tyrosine kinase 3 (flt3)-activated bone marrow (BM) cells for the treatment of hSOD1(G93A) transgenic mice. Motor function and survival showed greater improvement in the SCF group than in the group receiving BM cells that had not been activated (BMT alone group), although no improvement was shown in the flt3 group. In addition, larger numbers of BM-derived cells that expressed the microglia marker Iba1 migrated to the spinal cords of recipient mice compared with the BMT alone group. Moreover, after SCF activation, but not flt3 activation or no activation, the migrating microglia expressed glutamate transporter-1 (GLT-1). In spinal cords in the SCF group, inflammatory cytokines tumor necrosis factor-α and interleukin-1ß were suppressed and the neuroprotective molecule insulin-like growth factor-1 increased relative to nontreatment hSOD1(G93A) transgenic mice. Therefore, SCF activation changed the character of the migrating donor BM cells, which resulted in neuroprotective effects. These studies have identified SCF-activated BM cells as a potential new therapeutic agent for the treatment of ALS.

Gene therapy for neuropathic pain by silencing of TNF-α expression with lentiviral vectors targeting the dorsal root ganglion in mice.

Neuropathic pain can be a debilitating condition. Many types of drugs that have been used to treat neuropathic pain have only limited efficacy. Recent studies indicate that pro-inflammatory mediators including tumor necrosis factor α (TNF-α) are involved in the pathogenesis of neuropathic pain. In the present study, we engineered a gene therapy strategy to relieve neuropathic pain by silencing TNF-α expression in the dorsal root ganglion (DRG) using lentiviral vectors expressing TNF short hairpin RNA1-4 (LV-TNF-shRNA1-4) in mice. First, based on its efficacy in silencing TNF-α in vitro, we selected shRNA3 to construct LV-TNF-shRNA3 for in vivo study. We used L5 spinal nerve transection (SNT) mice as a neuropathic pain model. These animals were found to display up-regulated mRNA expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY), injury markers, and interleukin (IL)-6, an inflammatory cytokine in the ipsilateral L5 DRG. Injection of LV-TNF-shRNA3 onto the proximal transected site suppressed significantly the mRNA levels of ATF3, NPY and IL-6, reduced mechanical allodynia and neuronal cell death of DRG neurons. These results suggest that lentiviral-mediated silencing of TNF-α in DRG relieves neuropathic pain and reduces neuronal cell death, and may constitute a novel therapeutic option for neuropathic pain.

4-Hydroxy hexenal derived from docosahexaenoic acid protects endothelial cells via Nrf2 activation.

Recent studies have proposed that n-3 polyunsaturated fatty acids (n-3 PUFAs) have direct antioxidant and anti-inflammatory effects in vascular tissue, explaining their cardioprotective effects. However, the molecular mechanisms are not yet fully understood. We tested whether n-3 PUFAs showed antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcriptional factor for antioxidant genes. C57BL/6 or Nrf2(-/-) mice were fed a fish-oil diet for 3 weeks. Fish-oil diet significantly increased the expression of heme oxygenase-1 (HO-1), and endothelium-dependent vasodilation in the aorta of C57BL/6 mice, but not in the Nrf2(-/-) mice. Furthermore, we observed that 4-hydroxy hexenal (4-HHE), an end-product of n-3 PUFA peroxidation, was significantly increased in the aorta of C57BL/6 mice, accompanied by intra-aortic predominant increase in docosahexaenoic acid (DHA) rather than that in eicosapentaenoic acid (EPA). Human umbilical vein endothelial cells were incubated with DHA or EPA. We found that DHA, but not EPA, markedly increased intracellular 4-HHE, and nuclear expression and DNA binding of Nrf2. Both DHA and 4-HHE also increased the expressions of Nrf2 target genes including HO-1, and the siRNA of Nrf2 abolished these effects. Furthermore, DHA prevented oxidant-induced cellular damage or reactive oxygen species production, and these effects were disappeared by an HO-1 inhibitor or the siRNA of Nrf2. Thus, we found protective effects of DHA through Nrf2 activation in vascular tissue, accompanied by intra-vascular increases in 4-HHE, which may explain the mechanism of the cardioprotective effects of DHA.

Haematopoietic cells produce BDNF and regulate appetite upon migration to the hypothalamus.

Brain-derived neurotrophic factor (BDNF) suppresses food intake by acting on neurons in the hypothalamus. Here we show that BDNF-producing haematopoietic cells control appetite and energy balance by migrating to the hypothalamic paraventricular nucleus. These haematopoietic-derived paraventricular nucleus cells produce microglial markers and make direct contacts with neurons in response to feeding status. Mice with congenital BDNF deficiency, specifically in haematopoietic cells, develop hyperphagia, obesity and insulin resistance. These abnormalities are ameliorated by bone marrow transplantation with wild-type bone marrow cells. Furthermore, when injected into the third ventricle, wild-type bone marrow mononuclear cells home to the paraventricular nucleus and reverse the hyperphagia of BDNF-deficient mice. Our results suggest a novel mechanism of feeding control based on the production of BDNF by haematopoietic cells and highlight a potential new therapeutic route for the treatment of obesity.

High sodium intake is associated with masked hypertension in Japanese patients with type 2 diabetes and treated hypertension.

BACKGROUND: Knowledge regarding the association between dietary sodium intake and the incidence of masked hypertension is limited. METHODS: A total of 193 Japanese type 2 diabetic outpatients who had been treated with antihypertensive agents and with office blood pressures <140/90 mm Hg were recruited. Masked hypertension was defined as having office blood pressure <140/90 mm Hg and 24-h mean ambulatory blood pressure ≥130/80 mm Hg. The dietary sodium intake was estimated by measuring the 24-h urinary sodium excretion. RESULTS: Masked hypertension was found in 128 (66.3%) patients. An age- and sex-adjusted univariate logistic regression analysis showed that urinary albumin excretion, renin-angiotensin system inhibitor use, office systolic blood pressure, and amount of dietary sodium intake were significantly associated with masked hypertension. A multivariate logistic regression analysis also identified an older age, renin-angiotensin system inhibitor use, an office elevated systolic blood pressure, and high dietary sodium intake to be independently associated with masked hypertension. When compared with those who consumed a low salt diet (sodium <120 mEq/day), the odds ratio for the risk of exhibiting masked hypertension in patients who consumed a medium salt diet (sodium 120 to <200 mEq/day) or a high salt diet (sodium ≥200 mEq/day) were 5.3 (P < 0.001) and 12.6 (P < 0.001), respectively. CONCLUSIONS: Masked hypertension is a common feature in type 2 diabetic patients being treated for hypertension. The observed association with sodium intake raised the hypothesis that excessive sodium intake may play a part in the genesis of masked hypertension in these patients.

Arterial stiffness and renal impairment in non-proteinuric type 2 diabetic patients.

UNLABELLED: Aims/Introduction: Although increases in urinary protein excretion generally precede a decline in the glomerular filtration rate, non-proteinuric renal impairment is common in patients with diabetes. In the present study, we examined the relationship between indices of arterial stiffness and renal function in type 2 diabetic patients without proteinuria. METHODS: Blood sampling, 24-h urine collection, brachial-ankle pulse wave velocity, and 24-h ambulatory blood pressure monitoring were performed in type 2 diabetic patients without overt proteinuria. The ambulatory arterial stiffness index was calculated as (1 - the regression slope of diastolic/systolic ambulatory blood pressure). Estimated glomerular filtration rate (eGFR)was calculated using the simplified prediction equation proposed by the Japanese Society of Nephrology. RESULTS: Of 213 non-proteinuric patients with type 2 diabetes, 60 (28.2%) had a reduced eGFR (<60 mL/min per 1.73 m(2)). Although the urinary albumin excretion rate was significantly correlated with the eGFR, 34 of 152 patients with normoalbuminuria (22.4%) had a reduced eGFR. The eGFR was significantly and negatively correlated with the ambulatory arterial stiffness index and brachial-ankle pulse wave velocity, but not with 24-h pulse pressure. Multivariate analysis revealed that increased age and increased urinary albumin excretion were independently associated with decreased eGFR. In addition, the ambulatory arterial stiffness index, but not brachial-ankle pulse wave velocity, were found to be independently and significantly associated with eGFR. CONCLUSIONS: Ambulatory arterial stiffness index is a marker for increased risk of renal failure in non-proteinuric patients with type 2 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00146.x, 2012).

Inactivation of TNF-α ameliorates diabetic neuropathy in mice.

Tumor necrosis factor (TNF)-α is a potent proinflammatory cytokine involved in the pathogenesis of diabetic neuropathy. We inactivated TNF-α to determine if it is a valid therapeutic target for the treatment of diabetic neuropathy. We effected the inactivation in diabetic neuropathy using two approaches: by genetic inactivation of TNF-α (TNF-α(-/-) mice) or by neutralization of TNF-α protein using the monoclonal antibody infliximab. We induced diabetes using streptozotocin in wild-type and TNF-α(-/-) mice. We measured serum TNF-α concentration and the level of TNF-α mRNA in the dorsal root ganglion (DRG) and evaluated nerve function by a combination of motor (MNCV) and sensory (SNCV) nerve conduction velocities and tail flick test, as well as cytological analysis of intraepidermal nerve fiber density (IENFD) and immunostaining of DRG for NF-κB p65 serine-276 phosphorylated and cleaved caspase-3. Compared with nondiabetic mice, TNF-α(+/+) diabetic mice displayed significant impairments of MNCV, SNCV, tail flick test, and IENFD as well as increased expression of NF-κB p65 and cleaved caspase-3 in their DRG. In contrast, although nondiabetic TNF-α(-/-) mice showed mild abnormalities of IENFD under basal conditions, diabetic TNF-α(-/-) mice showed no evidence of abnormal nerve function tests compared with nondiabetic mice. A single injection of infliximab in diabetic TNF-α(+/+) mice led to suppression of the increased serum TNF-α and amelioration of the electrophysiological and biochemical deficits for at least 4 wk. Moreover, the increased TNF-α mRNA expression in diabetic DRG was also attenuated by infliximab, suggesting infliximab's effects may involve the local suppression of TNF-α. Infliximab, an agent currently in clinical use, is effective in targeting TNF-α action and expression and amelioration of diabetic neuropathy in mice.

Association between serum soluble TNFα receptors and renal dysfunction in type 2 diabetic patients without proteinuria.

AIM: The aim of our study was to investigate whether serum levels of soluble tumor necrosis factor α receptor (sTNFR) 1 and 2 are markers for renal dysfunction in type 2 diabetic patients without overt proteinuria. METHODS: Japanese type 2 diabetic patients without overt proteinuria (n = 168) enrolled in the prospective observational follow-up study in 2001 were retrospectively analyzed. At baseline, the serum levels of sTNFR1 and sTNFR2 were measured by sandwich ELISA. The associations between these markers and change in estimated glomerular filtration rate (eGFR) after 5 years were evaluated. RESULTS: The levels of sTNFR1 and sTNFR2 closely correlated. At baseline, sTNFR1 and sTNFR2 associated inversely with eGFR. After 5 years, patients with high level of both sTNFR1 and sTNFR2 showed a greater decline in eGFR (-13.8 ± 15.5% versus -8.5 ± 11.8%, P = 0.027) and a 4-fold higher risk for a GFR decline of ≥ 25% than those with high level of only one receptor or low level of both receptors. These associations were enhanced in diabetic women. CONCLUSIONS: The higher levels of sTNFR1 and sTNFR2 were associated with a greater decline in eGFR in type 2 diabetic patients without proteinuria, especially in diabetic women.

Presence and functional role of the rapidly activating delayed rectifier K(+) current in left and right atria of adult mice.

Repolarization of cardiac action potentials is regulated by several types of K(+) currents. The present study examined the presence and functional significance of rapid delayed rectifier (I(Kr)) in left and right atrial myocytes of mouse heart, using whole-cell patch-clamp method. The functional role of ultrarapid delayed rectifier (I(Kur)) in the repolarization was also examined by blocking with 4-aminopyridine (50 µM). The presence of I(Kr) was detected in left and right atrial myocytes as an E-4031 (5 µM)-sensitive current that exhibited relatively rapid activation during depolarization and half activation voltage of -17.5 and -17.4 mV for left and right atrial myocytes, respectively. The current density of I(Kr) was similar between left and right atria. The prolongation of action potential measured at 50% repolarization evoked by 4-aminopyridine was significantly larger in left than in right atrium, which appears to be consistent with the larger amplitude of I(Kur) in left atrium. On the other hand, the prolongation of action potential measured at 90% repolarization caused by E-4031 was significantly larger in right than in left atrium. The longer action potential of right atrium, which may result at least partly from smaller amplitude of I(Kur), is likely to enhance the functional significance of I(Kr) in repolarization process of right atrium, despite of similar magnitude of I(Kr) in left and right atria. Our data thus identifies I(Kr) in mouse atria and indicates the presence of functional interaction between I(Kr) and I(Kur) that potentially contributes to repolarization heterogeneity in left and right atria of mouse heart.

Paradoxical brain embolism induced by Mycoplasma pneumoniae infection with deep venous thrombus.

A 21-year-old man had sudden-onset right hemiplegia and aphasia with respiratory infection. A chest X-ray disclosed consolidation in both lungs and magnetic resonance imaging showed an embolism in the left middle cerebral artery. A pelvic computed tomography scan revealed deep venous thrombus in both femoral veins. Patent foramen ovale was detected by transesophageal echocardiogram. Antibodies to M. pneumoniae were highly elevated, and hypercoagulability was subsequently detected. This case suggests that the possible pathogenic mechanism for M. pneumoniae infection-related stroke might be paradoxical brain embolism with deep venous thrombus as a consequence of the hypercoagulability related to this infection.