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Peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1beta) is known to be a transcriptional coactivator for SREBP-1, the master regulator of hepatic lipogenesis. Here, we evaluated the role of PGC-1beta in the pathogenesis of fructose-induced insulin resistance by using an antisense oligonucletoide (ASO) to knockdown PGC-1beta in liver and adipose tissue. PGC-1beta ASO improved the metabolic phenotype induced by fructose feeding by reducing expression of SREBP-1 and downstream lipogenic genes in liver. PGC-1beta ASO also reversed hepatic insulin resistance induced by fructose in both basal and insulin-stimulated states. Furthermore, PGC-1beta ASO increased insulin-stimulated whole-body glucose disposal due to a threefold increase in glucose uptake in white adipose tissue. These data support an important role for PGC-1beta in the pathogenesis of fructose-induced insulin resistance and suggest that PGC-1beta inhibition may be a therapeutic target for treatment of NAFLD, hypertriglyceridemia, and insulin resistance associated with increased de novo lipogenesis.

OBJECTIVE—Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo. RESEARCH DESIGN AND METHODS—To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser302, Ser307, and Ser612 mutated to alanine (Tg IRS-1 Ser→Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo. RESULTS—Tg IRS-1 Ser→Ala mice were protected from fat-induced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser→Ala mice displayed a significant increase in insulin-stimulated IRS-1–associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates. CONCLUSIONS—These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fat-induced insulin resistance in skeletal muscle in vivo.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor in stimulating lipogenesis in the liver. Protein-tyrosine phosphatase 1B (PTP1B) induces SREBP-1 gene expression via protein phosphatase 2A (PP2A) activation. PTP1B is reported to be anchored on the endoplasmic reticulum (ER) via its C-terminal tail, and change in intracellular localization of PTP1B by C-terminal-truncation did not alter its inhibitory effects on insulin signaling. In this study, we investigated whether the change in intracellular localization of PTP1B could influence SREBP-1 gene expression. Overexpression of C-terminal truncated PTP1B (PTP1BdeltaCT) in rat Fao cells did not induce SREBP-1 gene expression. Furthermore, PTP1BdeltaCT failed to bind PP2A, resulting in impaired PP2A activation, whereas overexpression of wild-type PTP1B (PTP1BWT) associated with PP2A. Moreover, a membrane-targeted PTP1BDeltaCT activated PP2A with restored PP2A binding, despite the absence of its C-terminal region. Finally, overexpression of PTP1BdeltaCT into mouse primary cultured hepatocytes failed to enhance SREBP-1c mRNA, whereas membrane-targeted PTP1BdeltaCT led to enhanced SREBP-1c mRNA in hepatocytes as well as PTP1BWT. In conclusion, membrane localization of PTP1B is essential for PP2A activation, which is crucial for its enhancement of SREBP-1 gene expression.

A variation of the response to Sodium glucose co-transporter 2 (SGLT2) inhibitors with age has not been investigated in patients with diabetes. The aim of this study was to assess renal threshold of glucose (RTg) before and after administration of an SGLT2 inhibitor in young adult patients (≤40 years) and older adult patients (40 years) with type 2 diabetes (T2DM).Twenty Japanese patients with T2DM were enrolled. Baseline data were obtained on the first day and dapagliflozin (5 mg) was administered at 6:00 on the second day. Glucose excursions were assessed by continuous glucose monitoring and urine samples were collected every hour during the daytime (7:00 to 15:00) on both days. RTg was estimated from the regression line of the scatter plot of the hourly mean glucose concentrations.After a single dose of dapagliflozin, RTg decreased from 121.5 to 6.1 mg/dl in the young adult group and from 151.0 mg/dl to -15.8 mg/dl in the older group. After dapagliflozin, the slope of the regression line was significantly steeper in the young adult group.Dapagliflozin was more effective in young patients because they showed a larger response of urinary glucose excretion.

Many patients with type 2 diabetes mellitus(T2DM) do not achieve satisfactory glycemic control by monotherapy alone, and often require multiple oral hypoglycemic agents (OHAs). Combining OHAs with complementary mechanisms of action is fundamental to the management of T2DM. Fixed-dose combination therapy(FDC) offers a method of simplifying complex regimens. Efficacy and tolerability appear to be similar between FDC and treatment with individual agents. In addition, FDC can enhance adherence and improved adherence may result in improved glycemic control. Four FDC agents are available in Japan: pioglitazone-glimepiride, pioglitazone-metformin, pioglitazone-alogliptin, and voglibose-mitiglinide. In this review, the advantages and disadvantages of these four combinations are identified and discussed.

We have reported the association of variations in the activating protein-2beta (AP-2beta) transcription factor gene with type 2 diabetes. This gene was preferentially expressed in 3T3-L1 adipocytes in a differentiation stage-dependent manner, and preliminary experiments showed that subjects with the disease-susceptible allele showed stronger expression in adipose tissue than those without the susceptible allele. Thus, we overexpressed the AP-2beta gene in 3T3-L1 adipocytes to clarify whether AP-2beta might play a crucial role in the pathogenesis of type 2 diabetes through dysregulation of adipocyte function. In cells overexpressing AP-2beta, cells increased in size by accumulation of triglycerides accompanied by enhanced glucose uptake. On the contrary, suppression of AP-2beta expression by small interfering RNA inhibited glucose uptake. Enhancement of glucose uptake by AP-2beta overexpression was attenuated by inhibitors of phospholipase C (PLC) and atypical protein kinase Czeta/lambda (PKCzeta/lambda), but not by a phosphatidylinositol 3-kinase (PI3-K) inhibitor. Consistently, we found activation of PLC and atypical PKC, but not PI3-K, by AP-2beta expression. Furthermore, overexpression of PLCgamma enhanced glucose uptake, and this activation was inhibited by an atypical PKC inhibitor, suggesting that the enhanced glucose uptake may be mediated through PLC and atypical PKCzeta/lambda, but not PI3-K. Moreover, we observed the increased tyrosine phosphorylation of Grb2-associated binder-1 (Gab1) and its association with PLCgamma, indicating that Gab1 may be involved in AP-2beta-induced PLCgamma activation. Finally, AP-2beta overexpression was found to relate to the impaired insulin signaling. We propose that AP-2beta is a candidate gene for producing adipocyte hypertrophy and may relate to the abnormal characteristics of adipocytes observed in obesity.

To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at -468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between -453 to -480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1). loss of binding of unidentified proteins to the region between -453 to -480 bp of the SREBP-1c promoter and 2). impaired insulin stimulation of SREBP-1c promoter activity.

To clarify the mechanism of abnormalities in mitochondrial expression and function in diabetic rat heart, we have studied the transcriptional activities of mitochondrial DNA using isolated intact mitochondria from the heart of either diabetic or control rats. The transcriptional activity of cardiac mitochondria isolated from diabetic rats decreased to 40% of the control level (P0.01). Consistently, in the heart of diabetic rats, the content of cytochrome b mRNA encoded by mitochondrial DNA was reduced to 50% of control (P0.01). This abnormal transcriptional activity of mitochondrial DNA could not be explained by mRNA or protein contents of mitochondrial transcription factor (mtTFA), but mtTFA binding to the promoter sequence of mitochondrial DNA, assessed by gel-shift assay, was attenuated in diabetic rats. In contrast, the mRNA expression of nuclear-encoded mitochondrial genes, such as ATP synthase-beta, was not affected by diabetes. Although O(2) consumption of the mitochondria from diabetic rats was decreased, H(2)O(2) production in these rats was increased compared with the control. Insulin treatment reversed all the abnormalities found in diabetic rats. These results clearly indicate that an impairment of binding activity of mtTFA to the promoter sequence has a key role in the abnormal mitochondrial gene expression, which might explain the mitochondrial dysfunction found in diabetic heart.

We evaluated the effect of sitagliptin on glycemic control, endogenous insulin secretion, and beta cell function in Japanese patients with type 2 diabetes mellitus (T2DM) receiving a combination of oral antidiabetics and basal insulin analog glargine (basal-supported oral therapy [BOT]). Twenty-one patients showing inadequate glycemic control with BOT were given dipeptidylpeptidase-4 inhibitor (DPP-4I) sitagliptin at 50 mg/day for 12 weeks. Clinical markers of glycemic control, HbA1c, glycated albumin (GA), and 1,5-anhydroglucitol (1,5-AG), were measured before and 4 and 12 weeks after the start of sitagliptin. A 2-hour morning meal test was performed upon enrollment and at 12 weeks, and plasma glucose (PG), serum C-peptide, and plasma intact proinsulin (PI) were measured. HbA1c, GA, and 1,5-AG at 4 and 12 weeks were significantly improved over enrollment levels. The area under the PG concentration curve (AUC-PG) during the meal test at 12 weeks was significantly reduced (from 350 ± 17 mg ･ hr/dL before sitagliptin treatment to 338 ± 21 mg ･ hr/dL [mean ± SE], P < 0.05,); the AUC-C-peptide was unchanged (from 3.4 ± 0.4 ng ･ hr/mL to 3.6 ± 0.5 ng ･ hr/mL). However, both fasting and 2-hour PI/C-peptide ratios at 12 weeks were significantly decreased (from 13.3 ± 2.3 to 11.1 ± 2.0 [P < 0 .05] and from 9.5 ± 1.6 to 5.3 ± 0.9 [P < 0.01], respectively). Adding sitagliptin to BOT in Japanese T2DM patients appears to improve glycemic control without increasing endogenous insulin secretion and to reduce fasting and 2-hour postprandial PI/C-peptide ratios.

To evaluate the effect of the DPP-4 inhibitor sitagliptin on intrahepatic lipid (IHL) content and body fat in overweight Japanese patients with type 2 diabetes.A prospective, 24-week, single-center, open-label comparative study enrolled 20 Japanese patients with type 2 diabetes (male: 11, female: 9) with a BMI≥25 kg/m(2) or fatty liver. Subjects were randomly assigned to receive treatment with sitagliptin (25 mg titrated up to 50 mg: S) or glimepiride (0.5 mg titrated up to 1 mg: G). After starting each treatment, IHL and total fat mass were evaluated by (1)H-magnetic resonance spectroscopy ((1)H-MRS) and dual energy X-ray absorptiometry (DEXA), respectively at baseline and at 12 weeks and 24 weeks.After 24 weeks, HbA1c levels showed a similar significant decrease in both groups from 7.2 (7.0, 7.5) to 6.6 (6.4, 6.8)%, (54 (53, 56) to 48(47, 49) mmol/mol) with S and 7.3(6.8, 7.4) to 6.6 (6.3, 6.7)%, (55 (51, 56) to 48 (46, 49) mmol/mol) with G, median (interquartile range), p0.05 vs. baseline, with no significant differences between the two groups. The IHL and total body fat mass were decreased in S group from 24.5(18.9, 36.6) to 20.5 (14.6, 28.5)% (p=0.009) and 22.5 (20.6, 33.7) to 21.6 (19.7, 32.4)kg (p=0.028), respectively, but not in G group.Our findings indicate that sitagliptin and glimepiride achieved similar glycemic control, but only sitagliptin reduced IHL and total body fat (UMIN: 000013356).

Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63+/-10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61+/-16% vs. 13+/-15% at 20 min, and 80+/-8% vs. 41+/-12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS- 1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.

We present an unusual case of clear cell carcinoma arising from endometriosis in a 43-year-old woman. During laparotomy, a right adnexal tumor was found to be a sigmoidal subserosal tumor. Concomitant abnormal findings were endometriosis externa and enlarged ovaries. Intraoperative cytology suggested an adenocarcinoma; however, because of the unusual nature of the tumor and because the patient had not been fully informed about colostomy, further surgery was postponed. During the postoperative follow-up, the tumor grew and repeat aspiration cytology also suggested an adenocarcinoma. The patient again underwent laparotomy, and a Miles operation, total abdominal hysterectomy, bilateral salpingo-oophorectomy, regional lymphadenectomy and descending colostomy were performed.

The postprandial glucose (PPG) level is reduced by α-GIs, glinides and DPP4Is through different pharmacological actions. The aim of this study was to compare the effect of sitagliptin (S) versus that of the combination of mitiglinide and voglibose (M+V) on markers of glycemic control.A randomized cross-over trial was performed in 20 patients with drug-naïve type 2 diabetes. The patients were randomized to receive S (50 mg/day) or M+V (1 tablet 3 times daily). Treatment was continued for 8 weeks, after which they were switched to the other regimen and treated for another 8 weeks. At baseline, after the first regimen, and after the second regimen, a meal test was performed.The markers of glycemic control were examined.Reduction of glucose excursion was significantly greater with M+V than with S. HbA1c did not change with either regimen. However, 1,5-anhydroglucitol showed a significant increase from baseline with both regimens (7.9 ± 4.3 μg/ml at baseline vs. 10.6 ± 5.5 with S, p0.05 and 15.1 ± 6.2 with M+V, p0.01). Compared with baseline, glycoalbumin was significantly reduced by M+V, but not S (19.6 ± 2.9% at baseline vs. 17.3 ± 3.8% with M+V, p0.05).M+V achieved better control of PPG excursion than S.

The aim of the present study was to evaluate the effect of insulin glargine (Gla) (as part of basal-supported oral therapy) on endogenous insulin secretion and beta-cell function in type 2 diabetic patients. In 33 insulin-naive patients showing poor glycemic control on treatment with sulfonylurea (SU)-based OADs without DPP4 inhibitors, once-daily injection of Gla was added without changing OADs, and the dose of Gla was titrated to attain a fasting plasma glucose (FPG)

Many diabetes patients who require insulin perform multiple subcutaneous injections every day that often cause pain, discomfort, and anxiety. We compared efficacy (glycemic control) and patient preference for two types of needle: a shorter straight needle (32 gauge×4 mm, straight wall; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan; hereafter referred to as BD32S4) and a thinner microtapered needle (33-gauge tip and 28-gauge base×5 mm, double-tapered wall; Terumo Corp., Tokyo, Japan; hereafter referred to as TR33T5) in a single-center study.Eighty-four patients with diabetes were enrolled in a randomized, open-label crossover trial. The patients injected their usual insulin dosage with one type of needle for 4 weeks and then switched to the other type for the next 4 weeks. The serum glycated albumin level was measured before and after each 4-week period. Each patient assessed pain during injection on a 150-mm visual analog scale (VAS). Needle preference, perceptions of handling, and acceptance were assessed by the patients, who completed a questionnaire after using each type of needle for 4 weeks.In total, 79 patients completed the study. There was no difference of glycemic control between the two needles. The mean VAS score was -14.5 mm (95% confidence interval, -20.9, -8.0 mm), indicating that the patients perceived less pain with the BD32S4 needle. In the overall evaluation, a significantly higher percentage of patients selected the BD32S4 as the better needle compared with the TR33T5 (60.3% vs. 19.2%; P0.0001).The BD32S4 needle was more highly evaluated and was preferred by the patients with respect to pain during injection, usability, and visual impression, without having a negative impact on glycemic control. The overall preference of patients for the shorter needle in this study suggests that needle length may be one of the major contributing factors for patients' comfort in insulin injection, although the other relevant factors of needles still need to be considered.

By 2030, nearly half of Americans will have nonalcoholic fatty liver disease. In part, this epidemic is fueled by the increasing consumption of caloric sweeteners coupled with an innate capacity to convert sugar into fat via hepatic de novo lipogenesis. In addition to serving as substrates, monosaccharides also increase the expression of key enzymes involved in de novo lipogenesis via the carbohydrate response element-binding protein (ChREBP). To determine whether ChREBP is a potential therapeutic target, we decreased hepatic expression of ChREBP with a specific antisense oligonucleotide (ASO) in male Sprague-Dawley rats fed either a high-fructose or high-fat diet. ChREBP ASO treatment decreased plasma triglyceride concentrations compared with control ASO treatment in both diet groups. The reduction was more pronounced in the fructose-fed group and attributed to decreased hepatic expression of ACC2, FAS, SCD1, and MTTP and a decrease in the rate of hepatic triglyceride secretion. This was associated with an increase in insulin-stimulated peripheral glucose uptake, as assessed by the hyperinsulinemic-euglycemic clamp. In contrast, ChREBP ASO did not alter hepatic lipid content or hepatic insulin sensitivity. Interestingly, fructose-fed rats treated with ChREBP ASO had increased plasma uric acid, alanine transaminase, and aspartate aminotransferase concentrations. This was associated with decreased expression of fructose aldolase and fructokinase, reminiscent of inherited disorders of fructose metabolism. In summary, these studies suggest that targeting ChREBP may prevent fructose-induced hypertriglyceridemia but without the improvements in hepatic steatosis and hepatic insulin responsiveness.

Strict postprandial glycemic control may have a preventive effect on atherogenesis in patients with type 2 diabetes. The α-glucosidase inhibitor (α-GI) miglitol is useful for controlling the early postprandial increase of glucose, but the combined effect of miglitol and multiple daily insulin injections (MDI) on glucose excursion has not been evaluated. First, we retrospectively compared the daily glucose profile, evaluated by self-monitoring of blood glucose (SMBG) at nine times on the day before discharge from hospital, between type 2 diabetic patients receiving MDI (n=81) or MDI plus miglitol at 150 mg daily (n=24). Second, we prospectively examined the effect of adding miglitol to MDI on the daily glucose profile (SMBG) in 19 other type 2 diabetic patients. Although the daily insulin dosage and the glucose level before meals did not differ between the two groups, the 1-h postprandial glucose level after each meal, 2-h glucose level after lunch and dinner, mean and standard deviation of glucose, and amplitude of glucose excursion were significantly lower or smaller in the MDI plus miglitol group than in the MDI group. All of these glucose parameters were significantly improved by adding miglitol to MDI in the prospective cohort of 19 patients. In conclusion, adding miglitol to MDI reduces postprandial glucose levels and attenuates daily glucose fluctuation in type 2 diabetic patients. This trial was registered with UMIN (no. UMIN000005383).

Insulin action is purportedly modulated by Drosophila tribbles homologue 3 (TRIB3), which in vitro prevents thymoma viral proto-oncogene (AKT) and peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. However, the physiological impact of TRIB3 action in vivo remains controversial. We investigated the role of TRIB3 in rats treated with either a control or Trib3 antisense oligonucleotide (ASO). Tissue-specific insulin sensitivity was assessed in vivo using a euglycaemic–hyperinsulinaemic clamp. A separate group was treated with the PPAR-γ antagonist bisphenol-A-diglycidyl ether (BADGE) to assess the role of PPAR-γ in mediating the response to Trib3 ASO. Trib3 ASO treatment specifically reduced Trib3 expression by 70% to 80% in liver and white adipose tissue. Fasting plasma glucose, insulin concentrations and basal rate of endogenous glucose production were unchanged. However, Trib3 ASO increased insulin-stimulated whole-body glucose uptake by ~50% during the euglycaemic–hyperinsulinaemic clamp. This was attributable to improved skeletal muscle glucose uptake. Despite the reduction of Trib3 expression, AKT2 activity was not increased. Trib3 ASO increased white adipose tissue mass by 70% and expression of Ppar-γ and its key target genes, raising the possibility that Trib3 ASO improves insulin sensitivity primarily in a PPAR-γ-dependent manner. Co-treatment with BADGE blunted the expansion of white adipose tissue and abrogated the insulin-sensitising effects of Trib3 ASO. Finally, Trib3 ASO also increased plasma HDL-cholesterol, a change that persisted with BADGE co-treatment. These data suggest that TRIB3 inhibition improves insulin sensitivity in vivo primarily in a PPAR-γ-dependent manner and without any change in AKT2 activity.

We evaluated whether hyperinsulinaemia stimulates the expression of transcription factor CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP-delta and leads to the induction of the chemokine (C-C motif) ligand 2 gene (Ccl2, also known as MCP-1) expression in aortas.Hyperinsulinaemia was induced by feeding rats a high-fructose diet. CCL2 production was analysed by ELISA. The expression of Ccl2, Cebpb and Cebpd mRNAs was investigated by quantitative RT-PCR. The binding of C/EBP-beta to Ccl2 was assessed by chromatin immunoprecipitation (ChIP) assay.Insulin at a concentration of 10 nmol/l significantly stimulated the expression of Cebpb, Cebpd and Ccl2 mRNAs, depending on activation of phosphatidylinositol 3-kinase (PI3K) in cultured vascular smooth muscle cells. The knock-down of C/EBP-beta with siRNA abolished the insulin-induced Ccl2 mRNA expression. In the aortas from fructose-fed rats, the levels of phosphorylation of Akt/protein kinase B, a downstream effector of PI3K, were also increased. The expression of Cebpb, Cebpd and Ccl2 mRNAs in the aortas from fructose-fed rats were significantly elevated, by 330, 300 and 300%, respectively, compared with those of control-fed rats. The induction Ccl2 mRNA expression in the aortas was significantly correlated with the expression of Cebpb and Cebpd mRNAs in the aortas. Furthermore, the ChIP assay showed elevated binding of C/EBP-beta to the 5' upstream region of Ccl2 in the aortas from fructose-fed rats.These findings clearly indicate the role of C/EBPs in the mechanism of upregulation of CCL2, an inflammation-related protein, observed in the hyperinsulinaemic state, which may initiate the process of atherosclerosis.

To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at -468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between -453 to -480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1). loss of binding of unidentified proteins to the region between -453 to -480 bp of the SREBP-1c promoter and 2). impaired insulin stimulation of SREBP-1c promoter activity.

To elucidate molecular mechanisms of high fructose-induced metabolic derangements and the influence of peroxisome proliferator-activated receptor-α (PPARα) activation on them, we examined the expression of sterol regulatory element binding protein-1 (SREBP-1) and PPARα as well as its nuclear activation and target gene expressions in the liver of high fructose-fed rats with or without treatment of fenofibrate. After 8-wk feeding of a diet high in fructose, the mRNA contents of PPARα protein and its activity and gene expressions of fatty acid oxidation enzymes were reduced. In contrast, the gene expressions of SREBP-1 and lipogenic enzymes in the liver were increased by high fructose feeding. Similar high fructose effects were also found in isolated hepatocytes exposed to 20 mM fructose in the media. The treatment of fenofibrate (30 mg · kg−1· day−1) significantly improved high fructose-induced metabolic derangements such as insulin resistance, hypertension, hyperlipidemia, and fat accumulation in the liver. Consistently, the decreased PPARα protein content, its activity, and its target gene expressions found in high fructose-fed rats were all improved by fenofibrate treatment. Furthermore, we also found that the copy number of mitochondrial DNA, the expressions of mitochondrial transcription factor A, ATPase-6 subunit, and uncoupling protein-3 were increased by fenofibrate treatment. These findings suggest that the metabolic syndrome in high fructose-fed rats is reversed by fenofibrate treatment, which is associated with the induction of enzyme expression related to β-oxidation and the enhancement of mitochondrial gene expression.

An improved procedure for the syntheses of the two isomeric 8-chloro (4) and 11-chlorobenzanthrone (6) was devised and their condensation, by Ullmann reaction, to the corresponding dibenzanthronyls was achieved with the object of synthesizing violanthronyl derivatives.The product (mp 118°C) of reduction of 1-chloroanthraquinone (2) by Sn and glacial acetic acid which had been reported to be 4-chloro-9-anthrone (5), was found, by chromatographical separation, to be mixed crystals of two isomeric (5) (mp 137°C) and 1-chloro-9-anthrone (3) (mp 118°C), whose ratio was about 2 : 1.(5) was converted into pure (6) (mp 178°C) in 46% yield by the glycerol condensation. On the other hand, (4) (mp 182°C) obtained from (3) in this manner was the same product as the one obtained by the glycerol condensation of the reduction product of (2) in cone. H2SO4 with Al powder. (2) was converted into isomeric chlorobenzanthrones by a simultaneous reaction of reduction with aniline sulfate and condensation with glycerol. The product was separated by alumina chlomatography to (6) in 18% yield and (4) in 15% yield.Ullmann reaction of (4) and (6) was carried out in naphthalene, and 95% yield of 8, 8'-dibenzanthronyl was obtained from the former, while 11, 11'-dibenzanthronyl was obtained only in 7.8% yield from the latter.

9, 9'-Dihalogeno-3, 3-dibenzanthronyls were prepared as intermediates for the synthesis of diviolanthronyl, a possible high molecular weight dyestuff or a possible electric semiconductor. They were fine yellow crystals and dissolved in concentrated sulfuric acid to give a dark red solution.The 9, 9'-dichloro derivative, mp 4425°C, of 3, 3'-dibenzanthronyl (6) was prepared by the dehydrogenation condensation of 9-chlorobenzanthrone in the presence of magnesium dioxide. 9-Chlorobenzanthrone was obtained by the condensation of α-naphthyl-m-chlorophenylketone in the presence of aluminum chloride or by the partial reduction of 4, 9-dichlorobenzanthrone.Though the 9, 9'-diiodo derivatve, mp 4168°C, of (6) was synthesized in a similar manner from 9-iodobenzanthrone, the reaction was extremely slow and the yield was only 18%. 9-iodo-benzanthrone, mp 1979°C, was prepared in 88% yield by the Griess reaction of 9-aminobenzanthrone in concentrated sulfuric acid.When (6) was treated with an excessive amount of bromine, 9, 9'-dibromo-3, 3'- dibenzanthronyl, mp 4414°C, was obtained in 64% yield. The product was identical with the authentic sample obtained by the dehydrogenation condensation of 9- bromobezanthrone. The 9, 9'-dibromo derivative was also prepared by the bromination of (6) in the presence of catalysts such as aluminum halides.

ポリプロピレン用染料を目的として, m-位にアルキル基( メチル, エチル, t-ブチル基) を有するp-ジスアゾべンゼンおよびナフタリンジスアゾベンゼン誘導体を合成した。ポリプロピレンの染色を非イオン活性剤を含む水分散浴で行ない, 淡および濃色の黄～ 橙色を得た。染色性には, 次の序列があった。メチル体> エチル体＞t-ブチル体＞ ナフタリン体染色繊維について,耐光,洗タク,およびドライ・クリーニング堅ロウ度試験を行なった。諸種堅ロウ度のうち, 耐光堅ロウ度は特に高く0.1% o. w .f.の極淡色においても, 優れた結果を得た。0.1% o.w . f .で,エチル体は6～7級,t-ブチル体は6＜ 級,ナフタリン体は6級であった。また,次の序列があった。t-ブチル体＞ エチル体〓 ナフタリン体〓 メチル体洗タク堅ロウ度には,次の序列があった。t-ブチル体〓 メチル体～ エチル体〓ナフタリン体ドライ・クリーニング堅ロウ度には,次の序列があった。メチル体～t-ブチル体〓エチル体＞ ナフタリン体

加熱による有機顔料の脱水と分解は顔料の物理,化学的性質および実用上の諸問題を考える上にきわめて重要であると思われる。ここでは各種のアゾスルホン酸塩において,塩を形成する無機金属および有機スルホン酸をそれぞれに変化させた試料の加熱による重量変化を測定して,脱水,分解と構造との関連を求めると次のごとくであった。(I)陽イオンの効果塩をつくる金属の種類のみが異なって相手が同一のアゾスルホン酸基である場合は,金属イオンの原子価/イオン半径の値が大きくなるにしたがって,脱水の終る温度(脱水点)は高くなり,急激な分解の始まる温度(分解点)は低くなり,また脱水点と分解点との間の緩慢な分解の度合および水和分子数は大きくなる。(II)陰イオンの効果塩をつくる金属が同一でアゾスルホン酸基の種類が異なった場合は,塩をつくる酸基(スルホン酸,カルボン酸基)の増すにしたがって,脱水点,分解点は高くなり,この間の緩慢な分解は大きくなる。また塩をつくるとつくらないとにかかわらず,アゾ基の両側に酸基のある構造は片側のみにあるものにくらべて,かなり熱に不安定である。同一の核内においてもその位置によって影響がある。(III)脱水点と水和分子数は水溶性に関係し,水和分子数,脱水点,脱水点と分解点との間の緩慢な分解の度合,分解点が加熱による変色に関係する。

酸アミドからアミドのカルボニル基を脱離させてアミンを合成する方法はホフマン反応として良く知られている。しかしこの方法はすべてのアミンの合成に適用できるというものではない。著者らの一人は先に改良ホフマン反応について報告したが,改良ホフマン反応においてはすべての試薬をメタノール溶液として用いるものであり,臭素もメタノール溶液として加える。本研究においてもこの改良ホフマン法の適用を検討し,その結果,従来法に比べて相当量の収率向上が認められた。すなわちn-カプリルアミド,n-ラウリルアミドからはそれぞれ対応するn-ヘプチルアミン,n-ウンデシルアミンが58.2%(従来法48%)および83.3%(78.3%)の収率で得られた。また芳香族アミンとしては,従来ホフマン反応ではできにくいとされていたオルタニル酸が25.4%(5.0%),3,5-ジブロム・カルボニルアミノフェノールが73.4%(41%)の収率で得られた。

ポリリン酸中で2,5-ビス(2-カルボキシアニリノ)-1,4-ベンゾキノン(I)の脱水閉環反応を行ない,鮮明な黄色のキノ[2,3-b]-アクリジン-6,7,13,14-(5H,12H)-テトロン(以下キナクリドン・キノン)(II)をIに対し74～76%の収率で得た。つぎに,5,12-ジヒドロ-キノ-(2,3-b)-アクリジン-7,14-ジオン(以下キナクリドン)(III)の合成を目的として,ポリリン酸とスズをもちいてIIを還元したが,この還元は5,6,12,13-テトラヒドロ-キノ-[2,3-b]アクリジン-7,14-ジオン(以下6,13-ジヒドロ・キナクリドン)(IV)を生成しやすく,還元生成物には多量に混在するので,これをアルカリ性アルコール中ニトロベンゼン-m-スルホン酸ナトリウムと処理して酸化し, IIに対し, 最高収率85%でIIIを得た。また,Iをポリリン酸中で閉環後,IIを単離せずにただちにスズを加えて還元し,ついで上と同様な酸化処理をほどこし,Iに対し82%の収率でIIIを得た。

メタクロレインをシアン化ナトリウム, ナトリウム, ナフタリン, トリブチルホスフィンその他で重合させた。トリフェニルホスフィンでは重合が起こらなかった。生成ポリマーの構造は開始剤の種類によって異なった。シアン化ナトリウムを用いた場合,-25℃ 以上の反応ではオレフィン重合が起こった。トリブチルホスフィンでは-78℃ でもオレフィン重合が起こった。カリウム,ナフタリンでは低温でカルボニル重合が主体となった。シアン化ナトリウムにより得られたポリメタクロレインを用いて, アセトフェノン, ベンジリデンアセトン, アセチルナフタリンおよびアセチルアントラセンとの反応を行なわせ, 不飽和ケトン構造を持つポリマーを合成した。ナトリウム, ナトリウムナフタリン,トリメチルベンジルアンモニウムヒドロキシドが触媒として有効に働いた。得られたポリマー不飽和ケトンは黄色固体で,可溶可融性であった。