Krebs Cycle Intermediates Protective against Oxidative Stress by Modulating the Level of Reactive Oxygen Species in Neuronal HT22 Cells.

Krebs cycle intermediates (KCIs) are reported to function as energy substrates in mitochondria and to exert antioxidants effects on the brain. The present study was designed to identify which KCIs are effective neuroprotective compounds against oxidative stress in neuronal cells. Here we found that pyruvate, oxaloacetate, and α-ketoglutarate, but not lactate, citrate, iso-citrate, succinate, fumarate, or malate, protected HT22 cells against hydrogen peroxide-mediated toxicity. These three intermediates reduced the production of hydrogen peroxide-activated reactive oxygen species, measured in terms of 2',7'-dichlorofluorescein diacetate fluorescence. In contrast, none of the KCIs-used at 1 mM-protected against cell death induced by high concentrations of glutamate-another type of oxidative stress-induced neuronal cell death. Because these protective KCIs did not have any toxic effects (at least up to 10 mM), they have potential use for therapeutic intervention against chronic neurodegenerative diseases.

Relationships between the risk of cardiovascular disease in type 2 diabetes patients and both visit-to-visit variability and time-to-effect differences in blood pressure.

AIMS: To determine whether visit-to-visit blood pressure (BP) variability can predict cardiovascular disease (CVD) incidence in type 2 diabetes patients independently of mean BP, and to analyze the time-to-effect relationship between BP and CVD risk. METHODS: We retrospectively enrolled 629 type 2 diabetes patients with no history of CVD who first visited our hospital between 1995 and 1996, made at least one hospital visit per year, were followed-up for at least 1 year, and had undergone four or more BP measurements. The patients were followed until June 2012 at the latest. RESULTS: CVD occurred in 66 patients. Variability in systolic or diastolic BP (SBP and DBP, respectively) was a significant predictor of CVD incidence, independent of mean SBP or DBP. CVD incidence was significantly associated with SBP during the preceding 3-5 years, with the highest risk occurring during the preceding 3 years. CONCLUSIONS: Visit-to-visit BP variability independently predicts CVD incidence in type 2 diabetes patients. Increased SBP over the preceding 3-5 years indicated a significant CVD risk. To prevent CVD, BP management should focus on stable and well-timed control. In particular, BP stabilization at an early phase and BP control during late phases are important.

Time-to-effect relationships between systolic blood pressure and the risks of nephropathy and retinopathy in patients with type 2 diabetes.

AIMS: To analyze time-to-effect relationships between systolic blood pressure (SBP) and the risks of development of nephropathy and retinopathy in patients with type 2 diabetes. METHODS: We retrospectively enrolled 647 patients with type 2 diabetes who first visited our hospital between 1995 and 1996, made ≥1 hospital visit per year, had been followed-up for ≥1year, and had undergone ≥4 SBP measurements. Of these, 352 with normoalbuminuria and 516 without retinopathy were followed through June 2012. RESULTS: Nephropathy developed in 90 patients and retinopathy in 113. Hazard ratios (HRs) for time-dependent SBP-associated nephropathy and retinopathy were the highest during 1year preceding each endpoint or censoring. The HRs for nephropathy had been steadily lower during the preceding 1-17 years, while that for retinopathy had been lower during the preceding 1-5 years and constant during the preceding 5-17 years. CONCLUSIONS: The time-to-effect relationship with SBP differed for the development of nephropathy and retinopathy. The long-term effect was obvious for nephropathy and borderline for retinopathy, while the short-term effect was stronger and evident for both. Continuous SBP lowering is necessary to prevent nephropathy, whereas SBP control during the preceding 5years seems to be important to prevent retinopathy.

Association between HbA1c variability and mortality in patients with type 2 diabetes.

AIMS: We aimed to evaluate the association between HbA1c variability and mortality due to all causes, cancer, and non-cancer in patients with type 2 diabetes independently of mean HbA1c levels. METHODS: We enrolled 754 patients with type 2 diabetes who first visited our hospital between 1995 and 1996, had been followed for at least 2years, and had undergone four or more HbA1c determinations. Patients were followed through June 2012. The standard deviation (SD) or coefficient of variation (CV) was used as a measure of HbA1c variability. Risk of death was evaluated by multivariate Cox proportional hazard models. RESULTS: Through June 2012, 63 patients died. Hazard ratios (HRs) for all-cause mortality and non-cancer mortality including cardiovascular diseases (CVD) increased across tertiles of both HbA1cSD and HbA1cCV. HRs for cancer mortality did not increase across tertiles of either HbA1cSD or HbA1cCV. Using a stepwise regression method, both HbA1cSD and HbA1cCV predicted all-cause mortality, especially non-cancer mortality. In contrast, mean HbA1c predicted cancer mortality. CONCLUSIONS: HbA1c variability is a predictor of all-cause mortality, especially non-cancer mortality including CVD, in patients with type 2 diabetes, independent of mean HbA1c level. In contrast, mean HbA1c, but not HbA1c variability, might predict cancer mortality.

Visit-to-visit variability in systolic blood pressure predicts development and progression of diabetic nephropathy, but not retinopathy, in patients with type 2 diabetes.

OBJECTIVE: To investigate whether visit-to-visit variability in systolic blood pressure (SBP) can predict development and progression of diabetic nephropathy and retinopathy in patients with type 2 diabetes mellitus (T2DM). METHODS: From 1995 through 1996, 664 T2DM patients visited our hospital for the first time and were subsequently examined 4 times or more and at least once annually. At first visit, 326 had normoalbuminuria, 644 had an estimated glomerular filtration rate (eGFR) of ≥ 45 ml/min/1.73 m(2), 526 had no diabetic retinopathy and 609 had no severe non-proliferative diabetic retinopathy (NPDR). They were followed through June 2012, at the latest. RESULTS: Ninety patients developed microalbuminuria, 76 showed decrease of eGFR to <45 ml/min/1.73 m(2), 113 developed mild-moderate NPDR and 50 progression to severe NPDR. The unadjusted, age- and sex-adjusted and multivariate-adjusted hazard ratios for development and progression of nephropathy, but not retinopathy, increased across tertiles of the standard deviation (SD) of SBP. Both the SD and coefficient of variation (CV) of SBP were significant predictors of development and progression of nephropathy, but not retinopathy, independently of mean SBP. CONCLUSION: Visit-to-visit SBP variability is an independent predictor of development and progression of diabetic nephropathy, but not retinopathy, in T2DM patients.

Effects of celiac superior mesenteric ganglionectomy on glucose homeostasis and hormonal changes during oral glucose tolerance testing in rats.

The liver plays an important role in maintaining glucose homeostasis in the body. In the prandial state, some of the glucose which is absorbed by the gastrointestinal tract is converted into glycogen and stored in the liver. In contrast, the liver produces glucose by glycogenolysis and gluconeogenesis while fasting. Thus, the liver contributes to maintaining blood glucose level within normoglycemic range. Glycogenesis and glycogenolysis are regulated by various mechanisms including hormones, the sympathetic and parasympathetic nervous systems and the hepatic glucose content. In this study, we examined a rat model in which the celiac superior mesenteric ganglion (CSMG) was resected. We attempted to elucidate how the celiac sympathetic nervous system is involved in regulating glucose homeostasis by assessing the effects of CSMG resection on glucose excursion during an oral glucose tolerance test, and by examining hepatic glycogen content and hepatic glycogen phosphorylase (GP) activity. On the oral glucose tolerance test, CSMG-resected rats demonstrated improved glucose tolerance and significantly increased GP activity compared with sham-operated rats, whereas there were no significant differences in insulin, glucagon or catecholamine levels between the 2 groups. These results suggest that the celiac sympathetic nervous system is involved in regulating the rate of glycogen consumption through GP activity. In conclusion, the examined rat model showed that the celiac sympathetic nervous system regulates hepatic glucose metabolism in conjunction with vagal nerve innervations and is a critical component in the maintenance of blood glucose homeostasis.

Efficacy and tolerability of rosiglitazone and pioglitazone in drug-naïve Japanese patients with type 2 diabetes mellitus: a double-blind, 28 weeks' treatment, comparative study.

OBJECTIVE: A 28-week, randomized, placebo-controlled study was performed to evaluate efficacy and tolerability of rosiglitazone in Japanese type 2 diabetes patients. RESEARCH AND DESIGN METHODS: 373 patients were randomized to rosiglitazone (4-8 mg/day), pioglitazone (15-45 mg/day) or placebo. Agents were titrated to maximum doses at fixed time points in a pre-defined manner. Primary endpoints were superiority of each active treatment compared to placebo in HbA(1c) at week 16, and non-inferiority between active agents in HbA(1c) at week 28, based on a -0.45% margin. RESULTS: At week 16, improvements versus placebo were observed with rosiglitazone 4 mg/day (-0.96%, p < 0.001) and pioglitazone 30 mg/day (-1.26%, p < 0.001). At week 28, rosiglitazone and pioglitazone were associated with significant changes from baseline of -0.94% and -1.35%, respectively and rosiglitazone produced statistically and clinically significant improvement versus placebo (-1.29%, CI: -1.62, -0.97). Pioglitazone also showed significant improvement versus placebo (-1.64%, CI: -1.96, -1.31). Non-inferiority of rosiglitazone (4-8 mg/day) to pioglitazone (30-45 mg/day) was not demonstrated (treatment-difference: -0.41%, 95% CI: -0.64, -0.18). More patients treated with pioglitazone were withdrawn from the study by adverse events compared with rosiglitazone (14 vs. 4, p = 0.015). Pioglitazone was associated with higher incidences of adverse events relating to edema and weight gain compared with rosiglitazone (edema: 25.2 vs. 11.3%, weight gain: 9.4 vs. 4.4%). There were no reports of ischemic heart disease or congestive heart failure in any treatment group. CONCLUSION: Although non-inferiority to pioglitazone up to 45 mg in efficacy was not shown, rosiglitazone was confirmed to have clinically meaningful efficacy over placebo and fewer fluid-related events than pioglitazone. The study is registered on ClinicalTrials.gov as protocol NCT00297063.

The effects of fasting plasma glucose variability and time-dependent glycemic control on the long-term risk of retinopathy in type 2 diabetic patients.

Long-term fasting plasma glucose (FPG) variability was a risk factor for proliferative diabetic retinopathy (PDR) independent of the mean FPG or HbA1c in people with type 2 diabetes. PDR development was also significantly associated with mean HbA1c more than 5 years earlier and with mean FPG more than 10 years earlier.

Efficacy and tolerability of vildagliptin as an add-on to glimepiride in Japanese patients with Type 2 diabetes mellitus.

AIM: To investigate the efficacy and tolerability of vildagliptin, a potent and selective dipeptidyl peptidase-4 inhibitor, as add-on to glimepiride in Japanese patients with Type 2 diabetes mellitus (T2DM) who were inadequately controlled. METHODS: This 12-week, randomized, double-blind, placebo-controlled study compared vildagliptin 50mg twice-daily (n=102) with placebo (n=100) when added to a stable dose of glimepiride (>or=1mg/d). RESULTS: Treatment groups were balanced at baseline (glycosylated hemoglobin [HbA(1c)], 7.9%; fasting plasma glucose, 163.8 mg/dL). During treatment HbA(1c) decreased progressively with vildagliptin, but remained unchanged with placebo. The adjusted mean change (AMDelta) at endpoint was -1.0+/-0.1 and -0.1+/-0.1% in vildagliptin- and placebo-treated patients (between-group Delta=-1.0+/-0.1%, P<0.001). A greater proportion of vildagliptin-treated patients had HbA(1c)

The effect of fasting plasma glucose variability on the risk of retinopathy in type 2 diabetic patients: retrospective long-term follow-up.

AIMS: This study aimed to determine whether fasting plasma glucose (FPG) variability can predict diabetic retinopathy development and progression independently of glycemic control. METHODS: Subjects consisted of 170 type 2 diabetes mellitus patients not showing diabetic retinopathy on their first visit to our hospital between 1966 and 1979, and continuously visited thereafter for 27-40 (mean, 33) years. Plasma glucose and HbA1c data obtained on every hospital visit were collected. As FPG variability parameter, standard deviations (SD) were used. RESULTS: The investigation in which patients were classified based on the median value of the mean HbA1c or FPG and that of FPG SD have revealed that the risk of retinopathy development and progression were similar between the group showing good control and high variability and that with poor control and low variability. In multivariate analysis models with the mean FPG or HbA1c as a covariate and models using the mean FPG or HbA1c as a time-dependent covariate after adjustment for age, sex, diabetes duration, BMI, hypertension, and hypoglycemia, FPG SD was a significant independent risk factor for retinopathy. CONCLUSION: In type 2 diabetic patients, FPG variability is a risk factor for diabetic retinopathy independent of the mean FPG or HbA1c.

Macrophage foam cell formation is augmented in serum from patients with diabetic angiopathy.

The differentiation of macrophages into cytokine-secreting foam cells plays a critical role in the development of diabetic angiopathy. J774.1, a murine macrophage cell line, reportedly differentiates into foam cells when incubated with oxidized LDL, ApoE-rich VLDL or WHHLMI (myocardial infarction-prone Watanabe heritable hyperlipidemic) rabbit serum. In this study, serum samples from Type 2 diabetic patients were added to the medium with J774.1 cells and the degree of foam cell induction was quantified by measuring lipid accumulation. These values were calculated relative to the activities of normal and WHHLMI rabbit sera as 0% and 100%, respectively, and termed the MMI (Macrophage Maturation Index). These MMI values reflected intracellular lipids, including cholesteryl ester assayed by GC/MS. Statistical analysis revealed MMI to correlate positively and independently with serum triglycerides, the state of diabetic retinopathy, nephropathy and obesity, but negatively with administration of alpha-glucosidase inhibitors or thiazolidinediones. Taken together, our results suggest that this novel assay may be applicable to the identification of patients at risk for rapidly progressive angiopathic disorders.

Fasting tests of insulin secretion and sensitivity predict future prediabetes in Japanese with normal glucose tolerance.

UNLABELLED: Aims/Introduction: Reduced insulin sensitivity and secretion are important in the pathogenesis of type 2 diabetes. Their relationships to prediabetes, impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) have been previously studied with the oral glucose tolerance test (OGTT). We investigated whether or not baseline measures of insulin secretion and sensitivity obtained from fasting blood specimens were related to the development of prediabetes and how these measures compared with those based on the OGTT. MATERIALS AND METHODS: In 152 Japanese subjects with normal glucose tolerance, we measured baseline plasma glucose and insulin after an overnight fast and during a 75 g OGTT, insulin resistance index (homeostasis model assessment [HOMA-IR]), and insulin secretion (insulinogenic index [30 min insulin - fasting insulin] ÷ [30 min glucose - fasting glucose] or HOMA-ß). RESULTS: At a 5-6 year (mean 5.7 years) follow-up examination, we confirmed 36 cases of prediabetes. After adjusting for age, sex, family history of diabetes, body mass index, and 2-h plasma glucose, the odds ratio comparing the lowest tertile (≤0.82) of insulinogenic index with the highest tertile (≥1.43) was 6.98 (95% confidence interval, 1.96-24.85) and was 10.72 (2.08-55.3) comparing the lowest tertile (≤76.3) of HOMA-ß with the highest tertile (≥122.1), whereas the respective odds ratios of HOMA-IR were 3.74 (1.03-13.57) and 10.89 (1.93-61.41) comparing the highest tertile (≥1.95) with the lowest tertile (≤1.25). CONCLUSIONS: Lower insulin secretion and sensitivity are independent risk factors for prediabetes. Clinically practical identification of those at risk for prediabetes is obtainable from HOMA-ß and HOMA-IR, both of which are measured in fasting state. (J Diabetes Invest, doi: 10.1111.j.2040-1124.2010.00041.x, 2010).

Effect of mitiglinide on glycemic control over 52 weeks in Japanese type 2 diabetic patients insufficiently controlled with pioglitazone monotherapy.

This study was performed to examine the efficacy and safety of the rapid- and short-acting insulinotropic SUR ligand mitiglinide given as add-on therapy for 52 weeks in type 2 diabetic patients whose blood glucose was insufficiently controlled by pioglitazone monotherapy. Type 2 diabetic patients aged > or = 20 years with postprandial plasma glucose (PPG1 or 2) > or = 200 mg/dL and glycated hemoglobin (HbA(1C)) 6.5-<9.0% despite receiving pioglitazone 15-45 mg/day were additionally treated with concomitant mitiglinide 10 mg tid p.o. for a total treatment period of 52 weeks. In 171 patients recruited, HbA(1C) was significantly reduced from 7.64 +/- 0.77% at baseline to 6.84 +/- 0.73%, 6.64 +/- 0.64%, 6.67 +/- 0.57% and 6.81 +/- 0.65% at weeks 16, 28, 40, and 52, respectively. Over half the patients achieved HbA(1C) target of <7.0%, and one third <6.5%. Significant reductions in fasting plasma glucose (FPG) and PPG 1 and 2 hours after a meal versus baseline were noted at all time-points evaluated. The most frequently noted adverse reactions were hypoglycemic symptoms, weight gain, and peripheral edema (all mild). In type 2 diabetic patients combination therapy with mitiglinide and pioglitazone exerted significant long-term improvements in HbA(1C), FPG, and PPG and was well tolerated. This drug combination therapy is a promising means of alleviating insufficient pancreatic insulin secretion and insulin resistance.

Addition of mitiglinide to pioglitazone monotherapy improves overall glycemic control in Japanese patients with type 2 diabetes: a randomized double blind trial.

A 16-week, multicenter, randomized, double blind, parallel-group study was performed to examine whether additional administration of mitiglinide improves glycemic control in Japanese type 2 diabetic patients who are insufficiently controlled by pioglitazone monotherapy. Japanese adult type 2 diabetic patients were at first treated with diet plus pioglitazone 15-30 mg/day for 4 weeks then randomized to receive additional mitiglinide 5 or 10 mg or placebo tid for a further 16 weeks. In all, 381 patients were randomized. At final evaluation, glycated hemoglobin (HbA(1C)) was reduced by (mean +/- SD) -0.02 +/- 0.60% in the pioglitazone monotherapy group and by -0.45 +/- 0.77% and -0.67 +/- 0.59% in the mitiglinide 5 and 10 mg combination groups, respectively (both p<0.001 vs. pioglitazone monotherapy group). The percentage of patients who achieved HbA(1C) targets was significantly (p<0.001) higher in the mitiglinide combination groups than in the pioglitazone monotherapy group. Significant improvements in fasting plasma glucose and postprandial plasma glucose were noted in the mitiglinide combination groups versus the pioglitazone monotherapy group. No increase in adverse events was noted when mitiglinide was administered concomitantly with pioglitazone monotherapy. Hypoglycemic adverse events were infrequently and similarly observed in all three groups. Body weight gain and edema presented no clinical problem. HbA(1C) was significantly improved in the mitiglinide combination groups compared with the pioglitazone monotherapy group, and significantly more patients achieved HbA(1C) targets. Therefore mitiglinide effectively improves glycemic control in type 2 diabetic patients who are inadequately controlled by pioglitazone monotherapy.

Vildagliptin dose-dependently improves glycemic control in Japanese patients with type 2 diabetes mellitus.

OBJECTIVE: To assess the efficacy and tolerability of vildagliptin (10, 25 or 50mg bid) in Japanese patients with type 2 diabetes mellitus (T2DM). METHODS: This 12-week, multicenter, randomized, double-blind, placebo-controlled, parallel-group study was performed in 291 patients. The primary assessment was change from baseline to endpoint in HbA1c. RESULTS: Baseline HbA1c averaged 7.4%, and the between-treatment difference (vildagliptin-placebo) in the HbA1c adjusted mean change was -0.8%, -1.0% and -1.2% with vildagliptin 10, 25 and 50mg bid, respectively (p<0.001). Relative to baseline, body weight did not change significantly in vildagliptin groups. There was no increase in incidence of adverse events in the vildagliptin groups (62.0%, 62.5% and 61.8%, 10, 25 and 50mg bid, respectively) compared to placebo (73.6%). No deaths or drug-related serious adverse events were reported. Seven hypoglycemic events were observed (four events (n=3), two events (n=2), and one event (n=1) in the vildagliptin 10 and 50mg bid, and placebo, respectively) and none of them were severe or dose related. CONCLUSION: Vildagliptin 50mg bid was considered to be the most effective and well-tolerated dose, and therefore can be considered the recommended clinical dose for Japanese patients with T2DM.

[Influence of plasma glucose variability and age on onset of diabetic retinopathy in diabetic patients analysis of results of long-term outpatient follow-up for 30 years or more].

AIM: Diabetes mellitus (DM) outpatients were followed for 30 years or more. The impact of age and variability of fasting plasma glucose (FPG) and HbA(1C) on the onset of simple diabetic retinopathy (SDR) was analyzed. METHODS: The analysis included 84 DM patients who were free of retinopathy on their first visit between 1969 and 1977, then followed at the outpatients clinic through 2006. The plasma glucose and HbA(1C) were measured on every visit. The indices of variability were expressed as standard deviation (SD), coefficient of variance (CV) and range. RESULTS: The SDR incidence was significantly higher in the group of FPG SD > or =37 mg/dl (n=21) than in that of < 37 mg/dl (n=63). The hazard ratio, adjusted for the mean FPG, presence of hypoglycemia, age, duration of diabetes, hypertension and treatment of diabetes was 2.64 (95% CI: 1.26-5.50). The mean HbA(1C), HbA(1C) SD, mean FPG and FPG SD were significant risk factors for onset of SDR. Multivariate analysis identified the mean HbA(1C) and FPG SD as significant independent factors of increase in the risk of SDR onset. The SDR incidence was significantly lower in those aged 42 y or more (n=45) than in those under 42 y (n=39). The hazard ratio, adjusted for the mean FPG, gender, duration of diabetes, hypertension, and treatment of diabetes was 0.53 (95% CI: 0.30-0.95). All values for mean, SD and CV of FPG were significantly lower in the age group of > or = 42 y. CONCLUSIONS: The risk of SDR onset in DM patients increased with the mean values of HbA(1C) and/or FPG and also with the variability of these parameters. The risk decreased in the group above age 42, which was speculated to be due to the smaller variability in FPG and also due to the fact that subjects included in the group started SDR at the age exceeded the age of predilection for SDR onset.

Use of insulin glargine in Japanese patients with type 1 diabetes.

OBJECTIVE: To evaluate the results of treatment with an insulin glargine-based regimen as compared with those of an NPH insulin-based regimen. METHODS: We reviewed the charts of 83 Japanese patients with Type 1 diabetes treated with insulin glargine for 12 months. PATIENTS: Median age, 56.9 years (range, 24.6-74.8 years), mean (+/-S.D.) body mass index, 21.2 (+/-2.2) kg/m2. RESULTS: The average HbA1c level of the cohort was 7.8 +/- 1.2% at baseline and 7.7 +/- 1.0% at the end of the 12-month treatment (P=0.34). The average insulin requirement per day in the cohort remained unchanged after the 12-month treatment (35.0 +/- 11.6 units/day versus 35.2 +/- 11.2 units/day (P=0.58). Of the 36 patients who were receiving twice or three times daily injections of NPH insulin, 30 could be switched to a single-daily injection of insulin glargine. The frequency of severe hypoglycemia with unconsciousness became lower after switching to the insulin glargine-based regimen than during treatment with the NPH-based regimen. The average ratio of the daily usage of insulin glargine to that of total insulin after 12 months was smaller than that reported from other countries (0.34 +/- 0.09). CONCLUSION: These results obtained from a larger number of patients as compared to previous Japanese studies confirm earlier reports that insulin glargine provides equivalent glycemic control to human NPH insulin, with a lower incidence of severe hypoglycemia. Thus, treatment with insulin glargine provides some benefits to Japanese patients with Type 1 diabetes.

Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity.

AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.

A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis.

Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast two-hybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APE-induced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.