Postprandial Dynamics of Plasma Glucose,Insulin, and Glucagon in Patients with Type 2 Diabetes Treated with Saxagliptin Plus Dapagliflozin Add-On to Metformin Therapy

ObjectiveTo analyze changes in plasma glucose, insulin, and glucagon in relation to glycemic response during treatment with dual add-on of saxagliptin (SAXA) plus dapagliflozin (DAPA) to metformin XR (MET) compared with SAXA add-on or DAPA add-on alone to MET in patients with type 2 diabetes mellitus (T2DM) poorly controlled with MET.MethodsDouble-blind trial in adults with glycated hemoglobin (HbAlc) ≥ 8.0 to ≤ 12.0% randomized to SAXA 5 mg/day plus DAPA 10 mg/day (n = 179), or SAXA 5 mg/day and placebo (n = 176), or DAPA 10 mg/day and placebo (n = 179) added to background MET ≥ 1,500 mg/ day. The mean change from baseline in the area under the curve from 0 to 180 minutes (AUC_{0-180 min}) was calculated for glucose, insulin, and glucagon obtained during a liquid meal tolerance test (MTT).ResultsGlucose AUC_{0-180 min} an was reduced more from baseline with SAXA + DAPA + MET (-12,940 mg/dL) compared with SAXA + MET (-6,309 mg/dL) and DAPA + MET (-11,247 mg/dL). Insulin AUC_{0-180 min} significantly decreased with SAXA + DAPA + MET (-1,120 μU/mL) and DAPA + MET (-1,019 μU/mL) and increased with SAXA + MET (661 μU/mL). Glucagon AUC_{0-180 min} only increased with DAPA + MET (2,346 pg/mL). The changes in glucose (P < .0001) and insulin (P = .0003) AUC_{0-180 min} correlated with change in HbA1c, whereas the change in glucagon AUC_{0-180 min} min did not (P = .27).ConclusionsWhen added to background MET, the combination of SAXA + DAPA provided additional reductions in glucose AUC_{0-180 min} and HbA1c without the increase in insulin seen with SAXA and without the increase in glucagon seen with DAPA. Changes in insulin and glucose but not glucagon AUC_{0-180 min} correlated with change in HbA1c. (Endocr Pract. 2014;20:1187-1197)     

The triterpene,methyl-3β-hydroxylanosta-9,24-dien-21-oate (RA3), attenuates high glucose-induced oxidative damage and apoptosis by improving energy metabolism

BackgroundHyperglycemia-induced cardiovascular dysfunction has been linked to oxidative stress and accelerated apoptosis in the diabetic myocardium. While there is currently no treatment for diabetic cardiomyopathy (DCM), studies suggest that the combinational use of anti-hyperglycemic agents and triterpenes could be effective in alleviating DCM.HypothesisTo investigate the therapeutic effect of methyl-3β-hydroxylanosta-9,24-dien-21-oate (RA3), in the absence or presence of the anti-diabetic drug, metformin (MET), against hyperglycemia-induced cardiac injury using an in vitro H9c2 cell model.MethodsTo mimic a hyperglycemic state, H9c2 cells were exposed to high glucose (HG, 33 mM) for 24 h. Thereafter, the cells were treated with RA3 (1 μM), MET (1 μM) and the combination of MET (1 μM) plus RA3 (1 μM) for 24 h, to assess the treatments therapeutic effect.ResultsBiochemical analysis revealed that RA3, with or without MET, improves glucose uptake via insulin-dependent (IRS-1/PI3K/Akt signaling) and independent (AMPK) pathways whilst ameliorating the activity of antioxidant enzymes in the H9c2 cells. Mechanistically, RA3 was able to alleviate HG-stimulated oxidative stress through the inhibition of reactive oxygen species (ROS) and lipid peroxidation as well as the reduced expression of the PKC/NF-кB cascade through decreased intracellular lipid content. Subsequently, RA3 was able to mitigate HG-induced apoptosis by decreasing the activity of caspase 3/7 and DNA fragmentation in the cardiomyoblasts.ConclusionRA3, in the absence or presence of MET, demonstrated potent therapeutic properties against hyperglycemia-mediated cardiac damage and could be a suitable candidate in the prevention of DCM.     

Salicylate acutely stimulates 5′-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscles

Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5′-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL. Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr¹⁷² phosphorylation of the α subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPKα1 and AMPKα2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-d-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL.     

What explains the differences between centres in the European screening trial? A simulation study

BackgroundThe European Randomised study of Screening for Prostate Cancer (ERSPC) is a multicentre, randomised screening trial on men aged 55–69 years at baseline without known prostate cancer (PrCa) at randomisation to an intervention arm invited to screening or to a control arm. The ERSPC has shown a significant 21% reduction in PrCa mortality at 13 years of follow-up. The effect of screening appears to vary across centres, for which several explanations are possible. We set to assess if the apparent differences in PrCa mortality reduction between the centres can be explained by differences in screening protocols.MethodsWe examined the centre differences by developing a simulation model and estimated how alternative screening protocols would have affected PrCa mortality.ResultsOur results showed outcomes similar to those observed, when the results by centres were reproduced by simulating the screening regimens with PSA threshold of 3 versus 4 ng/ml, or screening interval of two versus four years. The findings suggest that the differences are only marginally attributable to the different screening protocols.ConclusionThe small screening impact in Finland was not explained by the differences in the screening protocols. A possible reason for it was the contamination of and the unexpectedly low PrCa mortality in the Finnish control arm.     

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

Higher serum sPD-L1 levels after radiotherapy indicate poor outcome in hepatocellular carcinoma patients

BackgroundOur preclinical research reveals that radiotherapy (RT) promoted PD-L1 upregulation in tumor tissues and that higher PD-L1 after RT worsened the prognosis through immunosuppression. We sought to validate our experimental results in clinical cohorts and promote clinical application.Patients and methodsIn cohort 1, formalin-fixed paraffin-embedded samples were obtained from 46 HCC patients, 23 of whom received preoperative RT and the other 23 received direct surgery. A prospectively collected database contained 122 HCC patients treated with liver RT were enrolled in cohort 2. Blood samples were taken a day before and two weeks after RT. Patients in cohort 2 were further divided into two groups, exploration (73 patients) and validation (49 patients) groups.ResultsIn cohort 1, RT increased the expression of PD-L1 in tumor tissues (p = 0.001), and PD-L1 levels were associated with decreased cytotoxic T-cell infiltration and a trend toward poor prognosis (p = 0.14). Moreover, PD-L1 expression in tumor tissue positively correlated with soluble (s) PD-L1 in serum (R = 0.421, p = 0.046). Then, in cohort 2, we revealed RT increased sPD-L1 in serum (p < 0.001), which was associated with the number of circulating CD8+ T cells (R = -0.24, p = 0.036), indicating poor survival. Furthermore, patients with higher rate of sPD-L1 increase after RT have better treatment response (p < 0.001), PFS (p = 0.032) and OS (p = 0.045).ConclusionHigher post-RT serum sPD-L1, which may potentiate immune suppression effects, indicates a poor prognosis for HCC patients treated with RT.     

Transitioning from conformal radiotherapy to intensity-modulated radiotherapy after radical prostatectomy: Clinical benefit,oncologic outcomes and incidence of gastrointestinal and urinary toxicities

AimThe purpose of this study was to review genitourinary (GU) and gastrointestinal (GI) toxicity associated with high-dose radiotherapy (RT) delivered with 3-dimensional conformal radiotherapy (3D-CRT) and intensity-modulated radiotherapy (IMRT) or volumetric arc therapy (VMAT) following radical prostatectomy (RP).BackgroundRP is a therapeutic option for the management of prostate cancer (PrCa). When assessing postoperative RT techniques for PrCa, the published literature focuses on patients treated with 2-dimensional conventional methods without reflecting the implementation of 3D-CRT, IMRT, or VMAT.Materials and methodsA total of 83 patients were included in this analysis; 30 patients received 3D-CRT, and 53 patients received IMRT/VMAT. Acute and late symptoms of the GU and lower GI tract were retrospectively graded according to the Radiation Therapy Oncology Group and the European Organization for Research and Treatment of Cancer radiation toxicity grading systems. The relapse failure-free rate and overall survival were also evaluated.ResultsThe rate of acute GU toxicity was 9.4% vs. 13.3% for the IMRT/VMAT and 3D-CRT groups (p = 0.583). The 5-year actuarial rates of late GI toxicity for IMRT/VMAT and 3D-CRT treatments were 1.9% and 6.7%, respectively. The rate of late GU toxicity for the IMRT/VMAT and 3D-CRT treatment groups was 7.5% and 16.6%, respectively (p = 0.199). We found no association between acute or late toxicity and the RT technique in univariate and multivariate analyses.ConclusionPostprostatectomy IMRT/VMAT and 3D-CRT achieved similar morbidity and cancer control outcomes. The clinical benefit of highly conformal techniques in this setting is unclear although formal analysis is needed.     

Effect of i.c.v. infusion of the alpha-MSH agonist MTII on meal patterns in male rats following nicotine withdrawal

The present study explored the role of endogenous alpha-MSH in the alteration of meal patterns induced by nicotine (NIC) withdrawal. Male Sprague Dawley rats bearing third ventricle cannulas were placed in computerized food intake monitors. On days 1-21, the rats were given 4 mg/kg/day of NIC or saline (SAL) in four equal i.p. doses during the dark period. NIC suppressed (P < 0.05) food intake only during the first week. The normalization of food intake occurred when the reduced meal size of the NIC injected rats was countered by an increase in meal number. Despite the normalization of 24-h food intake, body weight in NIC rats was decreased (P < 0.05) for 21 days. On day 22, the rats were divided into 4 groups (n's = 7-8 each) and injected into the third ventricle with various doses of the alpha-MSH agonist MTII or artificial cerebrospinal fluid (aCSF): SAL + aCSF, SAL + MTII, NIC + aCSF, NIC + MTII. Infusion of MTII (30 ng/rat) suppressed (P < 0.01) dark phase food intake in both groups, but the NIC + MTII group ate (P < 0.05) more than the SAL + MTII group. Meal number during the dark phase was suppressed by MTII, but the NIC + MTII group took significantly more meals that the SAL + MTII group. Infusion of MTII suppressed meal size in SAL and NIC treated rats, but this effect was attenuated in NIC treated rats. All meal parameters normalized by the day after i.c.v. infusion. These data indicate that NIC treatment differentially affects the neural controls of meal number and meal size and attenuates the suppression by MTII of meal number and meal size.     

噻唑衍生物WSF-SN-10激活AMPK的机制研究

目的:在噻唑衍生物中筛选新型AMPK激活剂并探究其激活AMPK的分子机制,以期找到副作用少的II型糖尿病治疗药物。方法:用14种噻唑衍生物处理293T细胞,用Western Blot筛选能显著提高AMPK磷酸化水平的化合物;用HPLC和FACS分别检测该化合物对细胞内AMP/ATP比值和Ca~(2+)浓度的影响,探究其激活AMPK的分子机制。结果:WSF-SN-10(2-(2-(6,6-二甲基双环[3,1,1]庚-2-亚基)肼基)-4-(4-氰基苯基)噻唑)为14种噻唑衍生物中活性最强的AMPK激活剂;20μM下WSF-SN-10激活AMPK的活性最强;用20μM WSF-SN-10处理293T细胞后,细胞内的AMP/ATP比值和LKB1的磷酸化水平分别上升至空白对照的1.94和3.04倍,同时Ca~(2+)浓度无明显变化,说明WSF-SN-10通过增加细胞内的AMP/ATP比值来激活AMPK。结论:噻唑衍生物WSF-SN-10能抑制细胞内的ATP合成来间接激活AMPK,是治疗II型糖尿病和肥胖症的潜在药物。     

Inverse planning in Gamma Knife radiosurgery: A comparative planning study

PurposeTo determine the advantages of inverse planning using a prerelease version of Leksell Gamma Knife® (LGK) Lightning (Elekta AB, Sweden) compared to manual forward planning.MethodsThirty-eight patients with metastases (MET, n = 15), vestibular schwannomas (VS, n = 11) and meningiomas (MEN, n = 12), treated with LGK Icon™ at our institution, were analyzed retrospectively. For each case, an inverse (inv) and inverse full coverage (fc) treatment plan was generated using LGK Lightning and compared to the clinical plans. Several dosimetry and efficiency characteristics were compared for each indication. The mean, median difference and interquartile range were reported and the significance was assessed with a paired-sample Wilcoxon test (significance level < 0.05). Further, the inter operator variability was analyzed for multiple users.ResultsInv and fc treatment plans show improved target coverage (up to 3.6%) for all analyzed paradigms. For inv plans, the selectivity is enhanced (MET: 2.9%; VS: 1.8%; MEN: 1%) and the organ at risk doses are significantly reduced (VS: up to 4.5%; MEN: up to 17.5%). For inv and fc plans, the beam on time (BOT) is shortened (MET: up to 7.9%; benign tumors: 49.5%). The inter operator variability analysis shows similar treatment plan quality with small differences in plan efficiency (difference in BOT: 1–3.3 min).ConclusionsLGK Lightning allows to generate improved LGK treatment plans regarding plan quality with reduced BOT compared to manual forward plans. The inter operator variability showed that multiple users with different experiences can generate similar treatment plan quality using LGK Lightning.     

Chromium enhances insulin responsiveness via AMPK

Trivalent chromium (Cr³⁺) is known to improve glucose homeostasis. Cr³⁺ has been shown to improve plasma membrane-based aspects of glucose transporter GLUT4 regulation and increase activity of the cellular energy sensor 5’ AMP-activated protein kinase (AMPK). However, the mechanism(s) by which Cr³⁺ improves insulin responsiveness and whether AMPK mediates this action is not known. In this study we tested if Cr³⁺ protected against physiological hyperinsulinemia-induced plasma membrane cholesterol accumulation, cortical filamentous actin (F-actin) loss and insulin resistance in L6 skeletal muscle myotubes. In addition, we performed mechanistic studies to test our hypothesis that AMPK mediates the effects of Cr³⁺ on GLUT4 and glucose transport regulation. Hyperinsulinemia-induced insulin-resistant L6 myotubes displayed excess membrane cholesterol and diminished cortical F-actin essential for effective glucose transport regulation. These membrane and cytoskeletal abnormalities were associated with defects in insulin-stimulated GLUT4 translocation and glucose transport. Supplementing the culture medium with pharmacologically relevant doses of Cr³⁺ in the picolinate form (CrPic) protected against membrane cholesterol accumulation, F-actin loss, GLUT4 dysregulation and glucose transport dysfunction. Insulin signaling was neither impaired by hyperinsulinemic conditions nor enhanced by CrPic, whereas CrPic increased AMPK signaling. Mechanistically, siRNA-mediated depletion of AMPK abolished the protective effects of CrPic against GLUT4 and glucose transport dysregulation. Together these findings suggest that the micronutrient Cr³⁺, via increasing AMPK activity, positively impacts skeletal muscle cell insulin sensitivity and glucose transport regulation.     

Subcutaneous administration of bovine superoxide dismutase protects lungs from radiation-induced lung injury

Abstract

Background. The objective of the present study was to determine whether single administration of the antioxidant enzyme bovine superoxide dismutase (bSOD) after radiation therapy (RT) mitigates development of pulmonary toxicity in rats. Methods. Female F344 rats (n = 60) were divided among six experimental groups: (1) RT, single dose of 21 Gy to the right hemithorax; (2) RT + 5 mg/kg bSOD; (3) RT + 15 mg/kg bSOD; (4) No RT; (5) sham RT + 5 mg/kg bSOD; and (6) sham RT + 15 mg/kg bSOD. A single subcutaneous injection of bSOD (5 or 15 mg/kg) was administered 24 h post-radiation. The effects of bSOD on radiation-induced lung injury were assessed by measurement of body weight, breathing frequency, and histopathological changes. Immunohistochemistry was used to evaluate oxidative stress (8-OHdG⁺, NOX4⁺, nitrotyrosine⁺, and 4HNE⁺ cells), macrophage activation (ED1⁺), and expression of profibrotic transforming growth factor-β or TGF-β in irradiated tissue. Results. Radiation led to an increase in all the evaluated parameters. Treatment with 15 mg/kg bSOD significantly decreased levels of all the evaluated parameters including tissue damage and breathing frequency starting 6 weeks post-radiation. Animals treated with 5 mg/kg bSOD trended toward a suppression of radiation-induced lung damage but did not reach statistical significance. Conclusions. The single application of bSOD (15 mg/kg) ameliorates radiation-induced lung injury through suppression of reactive oxygen species/reactive nitrogen species or ROS/RNS-dependent tissue damage.     

Anti‐aging pharmacology in cutaneous wound healing: effects of metformin,resveratrol, and rapamycin by local application

Cutaneous wounds are among the most common soft tissue injuries and are particularly hard to heal in aging. Caloric restriction (CR) is well documented to extend longevity; pharmacologically, profound rejuvenative effects of CR mimetics have been uncovered, especially metformin (MET), resveratrol (RSV), and rapamycin (RAPA). However, locally applied impacts and functional differences of these agents on wound healing remain to be established. Here, we discovered that chronic topical administration of MET and RSV, but not RAPA, accelerated wound healing with improved epidermis, hair follicles, and collagen deposition in young rodents, and MET exerted more profound effects. Furthermore, locally applied MET and RSV improved vascularization of the wound beds, which were attributed to stimulation of adenosine monophosphate‐activated protein kinase (AMPK) pathway, the key mediator of wound healing. Notably, in aged skin, AMPK pathway was inhibited, correlated with impaired vasculature and reduced healing ability. As therapeutic approaches, local treatments of MET and RSV prevented age‐related AMPK suppression and angiogenic inhibition in wound beds. Moreover, in aged rats, rejuvenative effects of topically applied MET and RSV on cell viability of wound beds were confirmed, of which MET showed more prominent anti‐aging effects. We further verified that only MET promoted wound healing and cutaneous integrity in aged skin. These findings clarified differential effects of CR‐based anti‐aging pharmacology in wound healing, identified critical angiogenic and rejuvenative mechanisms through AMPK pathway in both young and aged skin, and unraveled chronic local application of MET as the optimal and promising regenerative agent in treating cutaneous wound defects.     

Normal tissue complication probability models for severe acute radiological lung injury after radiotherapy for lung cancer

PurposeTo derive Normal Tissue Complication Probability (NTCP) models for severe patterns of early radiological radiation-induced lung injury (RRLI) in patients treated with radiotherapy (RT) for lung tumors. Second, derive threshold doses and optimal doses for prediction of RRLI to be used in differential diagnosis of tumor recurrence from RRLI during follow-up.Methods and materialsLyman-EUD (LEUD), Logit-EUD (LogEUD), relative seriality (RS) and critical volume (CV) NTCP models, with DVH corrected for fraction size, were used to model the presence of severe early RRLI in follow-up CTs. The models parameters, including α/β, were determined by fitting data from forty-five patients treated with IMRT for lung cancer. Models were assessed using Akaike information criterion (AIC) and area under receiver operating characteristic curve (AUC). Threshold doses for risk of RRLI and doses corresponding to the optimal point of the receiver operating characteristic (ROC) curve were determined.ResultsThe α/βs obtained with different models were 2.7–3.2 Gy. The thresholds and optimal doses curves were EUDs of 3.2–7.8 Gy and 15.2–18.1 Gy with LEUD, LogEUD and RS models, and μ_d of 0.013 and 0.071 with the CV model. NTCP models had AUCs significantly higher than 0.5. Occurrence and severity of RRLI were correlated with patients’ values of EUD and μ_d.ConclusionsThe models and dose levels derived can be used in differential diagnosis of tumor recurrence from RRLI in patients treated with RT. Cross validation is needed to prove prediction performance of the model outside the dataset from which it was derived.     

Complications after breast reconstruction with alloplastic material in breast cancer patients submitted or not to post mastectomy radiotherapy

Background and purposeBreast reconstruction following mastectomy is a relevant element of breast cancer treatment. The purpose of this study was to evaluate the influence of radiotherapy (RT) on local complications in patients with breast cancer that had undergone breast reconstruction with alloplastic material.Materials and methodsRetrospective study of breast cancer patients submitted to mastectomy and breast reconstruction from 2009 to 2013. Clinical and treatment variables were correlated with early and late complications.Results251 patients were included; mean age was 49.7 (25 to 78) years. Reconstruction was immediate in 94% of the patients, with 88% performed with a temporary tissue expander. Postoperative radiotherapy (RT) was delivered to 167 patients (66.5%). Early complications were present in 26.3% of the patients. Irradiated patients presented 5.4% incidence of late complications versus 2.4% for non-irradiated patients (p = 0.327). Diabetes (OR = 3.41 95% CI: 1.23–9.45, p = 0.018) and high body mass index (BMI) (OR = 2.65; 95% CI: 1.60–4.37, p < 0.0001) were the main risk factors. The overall incidence of late complications was 4.4%, with predominance of severe capsular contracture (8/11). Arterial hypertension (OR = 4.78; 95% CI: 1.97–11.63, p = 0.001), BMI (OR = 0.170; 95% CI: 0.048–0.607, p = 0.006) and implant placement (OR = 3.55; 95% CI: 1.26–9.99, p = 0.016) were related to late complications.ConclusionsThe overall rate of complications was low in this population. Radiotherapy delivery translated into a higher but not statistically significant risk of late complications when compared with the non-irradiated patients. Already well-known clinical risk factors for complications after breast reconstruction were identified.     

Analyses of putative anti-cancer potential of three STAT3 signaling inhibitory compounds derived from Salvia officinalis

The extract of Salvia officinalis (Common Sage) exhibited inhibitory activity of STAT3 signal after screening of several plants extracts using the STAT3-responsive reporter system. Cirsiliol, luteolin, and carnosol were identified from the methanol extract of Silvia officinalis as inhibitors of STAT3 signaling and the effects of these three compounds on STAT3 protein or growth inhibition on cancer cells was compared. Luteolin at the dose of 90 μM clearly suppressed the phosphorylation of STAT3 induced by IL-6, while carnosol was prone to decrease total STAT3 proteins at high doses (>90 μM). Cirsiliol had almost no effect. Since the three compounds exhibited similar concentration-dependent suppression patterns in the reporter assay except for cirsiliol became plateau beyond 30 μM, these compounds appeared to function as STAT3 inhibitory factors in different ways. The direct anti-proliferative activity of three compounds was examined with or without the anti-cancer drug gefitinib using HepG2 and A549 cells. The anti-proliferative effect of the three compounds was additively enhanced by gefitinib. At the doses of 3.6 μM, statistically significant suppression of proliferation was observed in HepG2 cells only by cirsiliol among the three compounds in the absence of gefitinib but all three compounds were prone to suppress the proliferation of HepG2 cells and A549 cells dose-dependently although cirsiliol showed a modest dose-dependency and this suppression of proliferation was enhanced by the addition of gefitinib. Cirsiliol, a dimethyoxylated flavone, activated the natural killer activity of KHYG-1 cells against erythroleukemia K562 cells like a hexamethoxylated flavone, nobiletin, suggesting that it may also have an indirect anti-cancer potential through activation of NK cells. These results shed light on the putative anti-cancer potential of Salvia officinalis.     

AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes

In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate ( [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} ). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions (>200 μM O₂) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . At physiologically relevant [O₂] (<40 μM O₂), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} with no change in sensitivity to [O₂] as assessed by estimates of cellular P₅₀ and P₉₀ values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} in normoxia. Treatment of hepatocytes with ≤200 μM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.     

Urolithin A suppresses glucolipotoxicity-induced ER stress and TXNIP/NLRP3/IL-1β inflammation signal in pancreatic β cells by regulating AMPK and autophagy

BackgroundPancreatic inflammation plays a key role in diabetes pathogenesis and progression. Urolithin A (UA), an intestinal flora metabolite of pomegranate, has anti-diabetic, anti-inflammatory and kidney protection effects among others. However, its effects on pancreatic inflammation and the potential mechanisms have not been clearly established.PurposeThis study aimed at investigating the molecular mechanisms of UA anti-pancreatic inflammation under a diabetic environment.MethodsDiabetes induction in male C57BL/6 mice was achieved by a high fat diet and intraperitoneal streptozotocin injections. Then, diabetic mice were orally administered with UA for 8 weeks. In vitro, endoplasmic reticulum stress and MIN6 pancreatic β cell inflammation were induced using 25 mM glucose and 0.5 mM palmitic acid. The effects of UA were evaluated by immunohistochemistry, Western blot, and enzyme linked immunosorbent assays. Finally, the underlying mechanisms were elucidated using an autophagy inhibitor (chloroquine, CQ) and an AMPK inhibitor (dorsomorphin dihydrochloride).ResultsUA significantly inhibited IL-1β secretion and TXNIP/NLRP3 expression in the pancreas of diabetic mice and in MIN6 pancreatic cells. UA downregulated the ER stress protein, p-PERK, and promoted AMPK phosphorylation. UA activated autophagy to inhibit TXNIP/NLRP3 IL-1β inflammatory signal, an effect that was reversed by CQ. Dorsomorphin 2HCL, reversed the autophagy-activation and anti-inflammatory effects of UA. Verapamil, clinically applied as an antiarrhythmic drug, is a TXNIP inhibitor for prevention of beta cell loss and diabetes development, but limited by its cardiac toxicity. In this study, verapamil (as positive control) inhibited NLRP3 /IL-1β signaling in MIN6 cells. Inhibitory effects of UA on TXNIP and IL-1β were weaker than those of verapamil (both at 50 μM, p < 0.05, p < 0.01). Conversely, inhibitory effects of UA on p62 were stronger, relative to those of verapamil (p < 0.05), and there were no differences in AMPK activation and LC3 enhancement effects between UA and verapamil.ConclusionUA is a potential anti-pancreatic inflammation agent that activates AMPK and autophagy to inhibit endoplasmic reticulum stress associated TXNIP/NLRP3/IL-1β signal pathway.     

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

BackgroundCa²⁺/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca²⁺-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear.MethodsAnti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ.ResultsOur data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca²⁺/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme.ConclusioncAMP/PKA signaling may confer Ca²⁺-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells.General significanceOur results suggest a novel cross-talk between cAMP/PKA and Ca²⁺/CaM/CaMKKβ signaling through regulatory phosphorylation.