A Randomized Trial of Step-up Treatment with Premixed Insulin Lispro-50/50 vs. Aspart-70/30 in Patients with Type 2 Diabetes Mellitus.

INTRODUCTION: When insulin treatment is started in patients with type 2 diabetes mellitus (T2DM), there are many regimens that control serum glucose levels to a normal range. Basal-bolus insulin therapy is one of the most effective treatments for improving glycemic control to prevent the progression of diabetic microvascular complications. This study was conducted to determine whether step-up insulin treatment with premixed insulin aspart-30/70 (BIAsp 30) or lispro-50/50 (Mix50) in Japanese patients with type 2 diabetes mellitus could achieve better glycemic control. METHODS: In this open label study, 72 insulin-naïve patients with poorly controlled T2DM (HbA1c ≥8.4%), who had been taking oral antidiabetic drugs for at least 12 months, were randomized to receive BIAsp 30 or Mix50 therapy. Patients started treatment of a pre-dinner injection of each type of insulin (Step 1). At 16 ± 2 weeks, a pre-breakfast injection of each type of insulin was added if HbA1c exceeded 7.4% (step 2). If patients had still not achieved HbA1c <7.4% after an additional 16 ± 2 weeks, a pre-lunch insulin injection was added (step 3). Hypoglycemic episodes were also recorded. RESULTS: The cumulative percentages of subjects who achieved HbA1c <7.4% were 36.1% (13/36) for both Mix50 and BIAsp 30 in step 1, 62.9% (23/36) for BIAsp 30 and 52.8% (19/36) for Mix50 in step 2, and 66.7% (24/36) in BIAsp 30 and 72.2% (26/36) in Mix50 in step 3. The achievement rates of HbA1c <7.4% were not statistically different between the two groups. A total of ten hypoglycemic episodes occurred in this study. However, there were no severe hypoglycemic episodes. All cases recovered by taking glucose and drinking juice. CONCLUSION: Mix50 step-up treatment has a clinical effect in achieving good glycemic control equal to that of BIAsp 30 treatment.

Improvement of glycemic control by re-education in insulin injection technique in patients with diabetes mellitus.

INTRODUCTION: The aim of this study was to evaluate the effectiveness of re-education in the insulin injection technique for glycemic control. METHODS: A preliminary experimental study was performed with 87 insulin-treated diabetic outpatients (11 with type 1 diabetes, 76 with type 2 diabetes; 43 men, 44 women). All patients had been treated with insulin for more than 3 years. After answering questions about the insulin injection technique, the patients' knowledge levels were scored. Correct answers and explanation sheets were subsequently given to all patients. The physicians in charge gave a short lecture and provided 10 min of individual advice. Two, three, and four months after re-education the HbA1c and glycoalbumin levels were measured. RESULTS: The mean HbA1c levels of almost all patients significantly improved from 7.46 ± 0.09% to 6.73 ± 0.10% (P < 0.01), and the mean glycoalbumin levels significantly improved from 22.76 ± 0.50% to 20.26 ± 0.68% (P < 0.01). Twenty-five patients demonstrated a poor understanding (score of ≤6 points) and showed a significant decrease in the HbA1c level from 7.62 ± 0.20% to 6.71 ± 0.21% (P = 0.02). Forty-three patients demonstrated a moderate understanding (score of 7 or 8 points) and showed a decrease in the HbA1c level from 7.40 ± 0.13% to 6.68 ± 0.07% (P = 0.07). Finally, 19 patients demonstrated a good understanding (score of ≥9 points) and showed a slight decrease in the HbA1c level from 7.38 ± 0.15% to 6.93 ± 0.12% (P = 0.09). Patients with a poor understanding showed the largest decrease in the mean level of HbA1c. CONCLUSION: Re-education in the insulin injection technique led to an improvement in glycemic control in insulin-treated diabetic patients, especially in those with a poor understanding of the insulin injection technique. More attention should be paid to these strategies for outpatients.

Severe hypoglycemia accompanied with thyroid crisis.

We report a 32-year-old Japanese women with severe hypoglycemia accompanied with thyroid crisis. She complained of dyspnea, general fatigue, and leg edema. She was diagnosed with hyperthyroidism with congestive heart failure and liver dysfunction. Soon after admission, sudden cardiopulmonary arrest occurred. She was then transferred to the intensive care unit. Her serum glucose level was 7 mg/dl. Intravenous glucose, hydrocortisone, diuretics, and continuous hemodiafiltration (CHDF) saved her. We considered that hypoglycemia occurred due to heart failure and liver dysfunction due to thyroid crisis.

Diagnostic criteria, clinical features, and incidence of thyroid storm based on nationwide surveys.

BACKGROUND: Thyroid storm (TS) is life threatening. Its incidence is poorly defined, few series are available, and population-based diagnostic criteria have not been established. We surveyed TS in Japan, defined its characteristics, and formulated diagnostic criteria, FINAL-CRITERIA1 and FINAL-CRITERIA2, for two grades of TS, TS1, and TS2 respectively. METHODS: We first developed diagnostic criteria based on 99 patients in the literature and 7 of our patients (LIT-CRITERIA1 for TS1 and LIT-CRITERIA2 for TS2). Thyrotoxicosis was a prerequisite for TS1 and TS2 as well as for combinations of the central nervous system manifestations, fever, tachycardia, congestive heart failure (CHF), and gastrointestinal (GI)/hepatic disturbances. We then conducted initial and follow-up surveys from 2004 through 2008, targeting all hospitals in Japan, with an eight-layered random extraction selection process to obtain and verify information on patients who met LIT-CRITERIA1 and LIT-CRITERIA2. RESULTS: We identified 282 patients with TS1 and 74 patients with TS2. Based on these data and information from the Ministry of Health, Labor, and Welfare of Japan, we estimated the incidence of TS in hospitalized patients in Japan to be 0.20 per 100,000 per year. Serum-free thyroxine and free triiodothyroine concentrations were similar among patients with TS in the literature, Japanese patients with TS1 or TS2, and a group of patients with thyrotoxicosis without TS (Tox-NoTS). The mortality rate was 11.0% in TS1, 9.5% in TS2, and 0% in Tox-NoTS patients. Multiple organ failure was the most common cause of death in TS1 and TS2, followed by CHF, respiratory failure, arrhythmia, disseminated intravascular coagulation, GI perforation, hypoxic brain syndrome, and sepsis. Glasgow Coma Scale results and blood urea nitrogen (BUN) were associated with irreversible damages in 22 survivors. The only change in our final diagnostic criteria for TS as compared with our initial criteria related to serum bilirubin concentration >3 mg/dL. CONCLUSIONS: TS is still a life-threatening disorder with more than 10% mortality in Japan. We present newly formulated diagnostic criteria for TS and clarify its clinical features, prognosis, and incidence based on nationwide surveys in Japan. This information will help diagnose TS and in understanding the factors contributing to mortality and irreversible complications.

A case of myeloma with hypercalcemia caused by high serum concentrations of both parathyroid hormone-related peptide (PTHrP) and macrophage inflammatory protein-1α (MIP-1α).

A 62-year-old woman was admitted with dry mouth, general fatigue, and severe back pain. Biochemistry examination showed extreme hypercalcemia (21.2 mg/dL). Bone marrow examination was negative, but needle biopsy of a metastatic lung tumor revealed abnormal plasma cells; thus, multiple myeloma stage III-A was finally diagnosed. Serum concentrations of both parathyroid hormone-related peptide (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) were markedly elevated (PTHrP 7.2 pmol/L, normal <1.1 pmol/L; MIP-1α 84.9 pg/mL, normal <46.9 pg/mL). Her myeloma appeared to have simultaneously caused two mechanisms producing hypercalcemia: humoral hypercalcemia of malignancy (HHM) by PTHrP and local osteolytic hypercalcemia (LOH) by MIP-1α. Therefore, the combination of two calcium-modulating abnormalities likely aggravated her hypercalcemia.

Cardiovascular complications of patients with aldosteronism associated with autonomous cortisol secretion.

CONTEXT: Primary aldosteronism (PA) is sometimes associated with the autonomous secretion of cortisol. OBJECTIVE: Our objective was to investigate the effect of autonomous cortisol secretion on the prevalence of cardiovascular events (CVE) in patients with PA. DESIGN: This was a retrospective cross-sectional study of cases collected from Gunma University Hospital between 2002 and 2010. PATIENTS: Seventy-six consecutive patients hospitalized for an evaluation of PA were analyzed. MAIN OUTCOME MEASURES: Rates of CVE dependent on autonomous cortisol secretion were examined. RESULTS: Of the 76 patients with PA, 21 (28%) had a history of CVE, including 14 with stroke, one with myocardial infarction, and six with atrial fibrillation. The multivariate logistic-regression and receiver operating characteristic analyses revealed that PA patients with CVE had significantly higher midnight cortisol levels than those without CVE; the adjusted odds ratio with a cutoff value of 7.4 µg/dl was 7.0 (95% confidence interval, 1.8-30.6; P = 0.006). In addition, results of the 1-mg dexamethasone suppression test with a cutoff value of 3.0 µg/dl differed significantly (odds ratio, 5.0; 95% confidence interval, 1.4-20.7; P = 0.018). Conversely, 67 and 50% of the PA patients with a midnight cortisol level of at least 7.4 µg/dl and 1-mg dexamethasone suppression test of at least 3.0 µg/dl had a history of CVE. Other factors such as age, expected glomerular filtration rate, blood pressure, glucose intolerance, the serum aldosterone concentration, plasma renin activity, and the duration of hypertension had no effect. CONCLUSION: The patients with PA associated with autonomous cortisol secretion had high incidence of CVE, and this association may further increase the risk of CVE in patients with PA.

A crossover study of rosuvastatin and pitavastatin in patients with type 2 diabetes.

INTRODUCTION: The effects of a low dose of rosuvastatin (ROS) and pitavastatin (PIT) on lipid profiles and inflammation markers were assessed in subjects with type 2 diabetes mellitus. METHODS: A total of 90 Japanese type 2 diabetes patients with hyperlipidemia (low-density lipoprotein cholesterol [LDL-C] ≥140 mg/dL) were enrolled in this study. They were randomly assigned to four groups with open-label treatment with ROS (2.5 mg daily) or PIT (2 mg daily); two groups were sequentially treated with both drugs, with crossover of medication after 12 weeks, and the other two groups underwent treatment with either ROS or PIT for 24 weeks. The primary endpoints were the percentage changes in LDL-C, high-density lipoprotein cholesterol (HDL-C) and triglyceride, and the LDL-C/HDL-C ratio. RESULTS: Both ROS and PIT lowered LDL-C and triglyceride, and increased HDL-C. In particular, significantly greater reduction in LDL-C was seen with ROS (-44.1%) than with PIT (-36.9%, P<0.01) in the crossover group from ROS to PIT, and the same result was detected in the crossover group from PIT (-34.8%) to ROS (-44.7%). The ratio of LDL-C/HDL-C was significantly reduced with ROS treatment (from 3.45 to 1.85) compared with that with PIT (from 3.45 to 2.22, P<0.01). Both ROS and PIT lowered plasma levels of high-sensitivity C-reactive protein (hsCRP), tumor necrosis factor (TNF)-alpha, and plasminogen activator inhibitor-1 (PAI-1). In addition, the hsCRP level with the administration of ROS was significantly improved compared with the administration of PIT. There was no significant correlation between changes in LDL-C and hsCRP, TNF-alpha, and PAI-1 levels. ROS and PIT did not have an adverse effect on glycemic control in type 2 diabetes patients. CONCLUSION: Therapy with both statins improved lipid profiles and reduced proinflammatory responses; however, 2.5 mg of ROS have a potent LDL-C-lowering and hsCRP-lowering effect compared with 2 mg of PIT in patients with diabetes.

Effects of twice-daily injections of premixed insulin analog on glycemic control in type 2 diabetic patients.

PURPOSE: Premixed insulin is effective to improve glycemic control; however, clinicians may be less likely to know which premixed insulin is appropriate for which patients. This study aimed to evaluate the effects of twice-daily injections of premixed insulin lispro on glycemic control in type 2 diabetic patients. MATERIALS AND METHODS: Forty type 2 diabetic patients, who had been treated with twice-daily injections of human protamine mixture 30/70 insulin for at least 12 months, were divided into two groups; one group whose blood glucose 2 hours after breakfast was greater than 200 mg/dL, was switched to lispro mix50, and the other group whose blood glucose 2 hours after breakfast < 200 was switched to lispro mix25. RESULTS: Glycated haemoglobin (HbA1c) significantly improved in the Mix50 group from 8.3% to 7.5% (at 12 weeks; p < 0.05), and to 7.5% (at 24 weeks; p < 0.05). On the other hand, HbA1c levels in the Mix25 group were slightly decreased from 8.1% to 7.7% at 12 weeks (p < 0.05), and to 7.9% at 24 weeks (not significant). Both postprandial plasma glucose and fasting plasma glucose levels were significantly improved in the Mix50 group, but not in the Mix25 group. Overall, 95% of subjects preferred premixed lispro insulin from human insulin in the viewpoint of the timing of insulin injection by questionnaire analysis. CONCLUSION: Switching from human protamine mixture 30/70 insulin to lispro mix50 twice-daily injection therapy in patients with high postprandial plasma glucose could improve their glycemic control and quality of life.

Troglitazone, a ligand of peroxisome proliferator-activated receptor-{gamma}, stabilizes NUCB2 (Nesfatin) mRNA by activating the ERK1/2 pathway: isolation and characterization of the human NUCB2 gene.

We recently identified a novel satiety peptide, nesfatin-1, containing 82 amino acids derived from the precursor peptide, nucleobindin 2 (NUCB2), from a troglitazone (TZ)-induced cDNA library. We examined the molecular mechanism underlying TZ-induced NUCB2 mRNA expression. Although TZ induced the mRNA expression in HTB185 cells, a nuclear run-on assay revealed no significant change in the transcription of the gene. Surprisingly, HTB185 cells possessed no functional peroxisome proliferator-activated receptor-gamma. We therefore examined the effect of TZ on the mRNA's stability. The half-life of NUCB2 mRNA was approximately 6 h, and incubation with TZ increased this to 27 h. Furthermore, this increase was completely inhibited by an ERK inhibitor, PD98059, and phosphorylated ERK1/2 was significantly increased after 30 min incubation with TZ. In addition, we cloned the entire NUCB2 gene and identified four adenylate/uridylate-rich elements (AREs) in the 3' untranslated region (UTR), to which several proteins of HTB185 extracts treated with TZ bound. The reporter assay fused with 3'UTR showed that the second and third AREs were crucial. Furthermore, the human NUCB2 gene spanned 55 kb and contained 14 exons and 13 introns. The transcriptional start site formed clusters around 246 bp upstream from the translational start site. We confirmed that a construct containing 5889 bp of the promoter region was very active in neuron-derived cell lines but not stimulated by TZ. These findings demonstrated a novel action of derivatives of thiazolidinediones, oral insulin-sensitizing antidiabetic agents, to stabilize the mRNA of NUCB2 through AREs in the 3'UTR by activating the ERK1/2 pathway independently of peroxisome proliferator-activated receptor-gamma.

Effect of raloxifene on serum lipids for type 2 diabetic menopausal women with or without statin treatment.

OBJECTIVE: Our aim was to investigate the effect of 1-year treatment with raloxifene, a selective estrogen receptor modulator, on plasma lipid profiles in Japanese postmenopausal type 2 diabetic patients. SUBJECTS AND METHODS: A total of 43 Japanese women with postmenopausal osteoporosis and type 2 diabetes with serum low-density lipoprotein cholesterol (LDL-C) <3.59 mmol/l, serum triglyceride <1.68 mmol/l and serum high-density lipoprotein cholesterol (HDL-C) >1.03 mmol/l, who took 60 mg/day of raloxifene for 12 months, were enrolled. For analysis, they were divided into 2 groups: nonhyperlipidemia (n = 23) and hyperlipidemia treated with statin (n = 20). RESULTS: Raloxifene treatment significantly induced a mean reduction in serum LDL-C from 2.90 to 2.36 and 2.67 mmol/l in the nonhyperlipidemia and statin-treated group, respectively. However, the reduction ratio of serum LDL-C showed a significant difference in the nonhyperlipidemia group (17%) compared to the statin-treated group (7%; p = 0.03). Although serum HDL-C showed an increase in both groups (from 1.45 to 1.58 vs. from 1.40 to 1.47 mmol/l), the increase ratio of serum HDL-C was not significant between the two groups. Raloxifene administration showed 15% reduction in the nonhyperlipidemia group (p = 0.02) and 13% reduction in the statin-treated group (p = 0.02) of urinary N-telopeptide of type I collagen. No significant change in blood HbA(1c) was observed in either group. CONCLUSION: The administration of raloxifene to type 2 diabetic women showed favorable efficacy on serum lipid profiles, particularly in patients without statin treatment.

A liver X receptor (LXR)-beta alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-beta.

We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-beta LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-beta gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-beta. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

Effects of changeover from voglibose to acarbose on postprandial triglycerides in type 2 diabetes mellitus patients.

INTRODUCTION: In this study, we examined the effects of the alpha-glucosidase inhibitors acarbose and voglibose on postprandial plasma glucose and serum triglyceride levels in patients with type 2 diabetes mellitus. METHODS: Twenty-one Japanese patients with type 2 diabetes were enrolled in this study. Subjects had been treated with voglibose for at least 3 months. They underwent a 400 kcal balanced food meal tolerance test before and 8 weeks after the changeover from voglibose to acarbose. Subjects were divided into two groups: the first group (low-dose group; n=11) was changed over from 0.6 mg/day voglibose to 150 mg/day acarbose, and the other (high-dose group; n=10) from 0.9 mg/day voglibose to 300 mg/day acarbose. RESULTS: The increment rate of postprandial plasma glucose ([plasma glucose 2 hours after test meal - fasting glucose]/fasting glucose) decreased from 34.7%+/-23.9% to 25.0%+/-24.6% (P=0.13) in the low-dose group, and decreased significantly from 56.1%+/-53.1% to 31.5%+/-36.0% (P=0.03) in the high-dose group after changeover. However, there were no significant changes in blood glycated hemoglobin (HbA(1c)) levels before and after changeover in either group. The increment rate of postprandial serum triglyceride (TG) ([serum TG 2 hours after test meal - fasting TG]/fasting TG) decreased significantly only in the high-dose group (52.4%+/-60.0% to 24.3%+/-16.6%) (P=0.05). No significant changes in serum high-density lipoprotein cholesterol levels were observed in either group, whereas serum low-density lipoprotein cholesterol levels decreased significantly from 3.20+/-0.25 to 2.65+/-0.18 mmol/L (P=0.04), only in the high-dose group. CONCLUSIONS: In patients with type 2 diabetes our findings suggest that acarbose 300 mg/day is superior to voglibose 0.9 mg/day in improving postprandial hyperglycemia and hypertriglyceridemia.

Carbohydrate response element binding protein gene expression is positively regulated by thyroid hormone.

The molecular mechanism of thyroid hormone (TH) effects to fatty acid metabolism in liver is yet to be clear. The carbohydrate response element-binding protein (ChREBP) as well as sterol response element-binding protein (SREBP)-1c plays a pivotal role in hepatic lipogenesis. Both SREBP-1c and ChREBP are target genes of liver X receptors (LXRs). Because LXRs and TH receptors (TRs) cross talk mutually in many aspects of transcription, we examined whether TRs regulate the mouse ChREBP gene expression. In the current study, we demonstrated that TH up-regulated mouse ChREBP mRNA and protein expression in liver. Run-on and luciferase assays showed that TH and TR-beta1 positively regulated the ChREBP gene transcription. The mouse ChREBP gene promoter contains two direct repeat-4 sites (LXRE1 and LXRE2) and EMSAs demonstrated that LXR-alpha and TR-beta1 prefer to bind LXRE1 and LXRE2, respectively. The direct repeat-4 deletion and LXRE2 mutants of the promoter deteriorate the positive regulation by TR-beta1, indicating that LXRE2 is functionally important for the regulation. We also showed that human ChREBP gene expression and promoter activities were up-regulated by TH. These data suggest that ChREBP mRNA expression is positively regulated by TR-beta1 and TH at the transcriptional level in mammals. This novel observation indicates that TH fine-tunes hepatic lipogenesis via regulating SREBP-1c and ChREBP gene expression reciprocally.

Tat-binding protein-1 (TBP-1), an ATPase of 19S regulatory particles of the 26S proteasome, enhances androgen receptor function in cooperation with TBP-1-interacting protein/Hop2.

The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

A novel splice variant of the nuclear coactivator p120 functions strongly for androgen receptor: characteristic expression in prostate disease.

We cloned a novel splicing variant for nuclear coactivator p120(alpha), designated as p120beta and studied its function and expression in several human prostate diseases. Transfection assays demonstrated that p120beta functions as a strong coactivator for androgen receptor (AR), but weakly for other nuclear receptors. GST-pull down assay showed that a glutamine-rich region of the p120 bound to the ligand-binding domain of AR. Interestingly, p120beta mRNAs were expressed predominantly in the normal prostate, androgen-responsive prostate cancers and an androgen-sensitive prostate cancer cell line, LNCaP, but weakly in recurrent cancers and the androgen-insensitive prostate cancer cell lines PC3 and DU145. Furthermore, knockdown of p120alpha by siRNA abolished coactivator activity on thyroid hormone receptors (TR) and PPARgamma, but did not affect that of ARs in PC3 cells. In addition, competitive assay with other nuclear receptors demonstrated that TR and PPARgamma did not inhibit p120beta-induced stimulation. These findings suggested that while p120alpha was essential for ligand-dependent stimulation of TRs and PPARgamma, p120beta acted as a coactivating protein predominantly for AR.

Herbal medicine, Hachimi-jio-gan, and its component cinnamomi cortex activate the peroxisome proliferator-activated receptor alpha in renal cells.

Hachimi-jio-gan is widely used to improve several disorders associated with diabetes, but its mechanism remains poorly understood. In an attempt to clarify the mechanism of Hachimi-jio-gan, we investigated the effects of this herbal medicine and its components in transfection studies of CV1 cells, especially nuclear receptor-mediated actions. One half (0.5) mg/ml of Hachimi-jio-gan activated peroxisome proliferator-activated receptor (PPARalpha), mediating the activation by 3.1-fold on DR1 response elements; however, it did not affect PPARgamma, thyroid hormone receptor, androgen receptor, estrogen receptor or RXR. In addition, this activation was observed in a dose-dependent manner. Next, to determine which components of Hachimi-jio-gan activate PPARalpha-mediated transcription, 8 of its components (rehmanniae radix, orni fructus, dioscoreae rhizoma, alismatis rhizoma, hoelen, moutan cortex, cinnamomi cortex, aconiti) were tested. Only cinnamomi cortex (1.0 mg/ml) increased PPARalpha-mediated transcription by 4.1-fold, and this activation was specific for PPAR alpha, and not for other nuclear receptors. Moreover, this PPARalpha-related activation by cinnamomi cortex is specifically observed in renal cells. Taken together, these findings indicate that Hachimi-jio-gan and cinnamomi cortex may have a pharmacological effect through the target site for PPARalpha.

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells.

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.

Tetrahydrobiopterin prevents endothelial dysfunction and restores adiponectin levels in rats.

Oxidative stress induces endothelial dysfunction and hypoadiponectinemia. We previously reported that supplementation with tetrahydrobiopterin (BH4), one of the most potent naturally occurring reducing agents and an essential cofactor of enzymatic NO synthase (NOS), ameliorates endothelial dysfunction and reverses hypoadiponectinemia as a result of oxidative stress in rats. To further confirm this hypothesis, we investigated the effects of treatment with BH4 on endothelium-dependent relaxation and adiponectin levels during oxidative stress in fructose-fed rats, which provide an animal model for the metabolic syndrome. Ingestion of a fructose diet for 8 weeks significantly impaired endothelium-dependent arterial relaxation in aortic strips and decreased plasma adiponectin levels, as well as adiponectin mRNA levels within adipose tissue. However, oral supplementation with BH4 (10 mg/kg day) over the final 4 weeks leads to a significant partial reversal of impaired endothelium-dependent arterial relaxation, as well as normalization of plasma adiponectin and fat adiponectin mRNA levels. Moreover, BH4 treatment of the fructose-fed rats significantly reduced the lipid peroxidation content of aorta, heart, liver, and kidney tissues, which were increased in fructose-fed rats. This effect of BH4 treatment may be due to its function as a cofactor for eNOS, as well as its anti-oxidative effects. Thus, BH4 might show promise for the treatment of oxidative stress-induced disorders, including the metabolic syndrome.

Identification of nesfatin-1 as a satiety molecule in the hypothalamus.

The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.

Correlation between serum osteoprotegerin and biomarkers of bone metabolism during anti-thyroid treatment in patients with Graves' disease.

Bone turnover is increased in favor of resorption in hyperthyroid patients. We aimed to examine whether osteoprotegerin (OPG), which has an inhibitory effect on osteoclasts, is correlated with any biomarkers for bone turnover in Graves' disease. Twenty-one patients with Graves' disease were examined in this study, before and after treatment. Briefly, OPG, calcium, phosphorus, PTH, free T3, free T4, TSH, TSH receptor antibody and TSH-stimulating antibody were measured. Elevated serum OPG levels were decreased in accordance with anti-thyroid treatment. This change of OPG level was associated with thyroid hormone free T4 (r = 0.175, p = 0.038) but not with free T3 (r = 0.164, p = 0.052) and TSH (r = 0.046, p = 0.59). Additionally, there was a negative correlation between OPG and PTH (r = -0.37, p = 0.0001). In stepwise regression analysis, the change in serum OPG levels during anti-thyroid treatment was significantly and independently associated with PTH (F ratio = 24.4, p < 0.0001). Our findings suggest that OPG may prevent excessive bone loss in the hyperthyroid state in accordance with the change of biomarkers for bone turnover.