Stimulation Of AMPK Research Articles

CASK Mediates Oxidative Stress-Induced Microglial Apoptosis-Inducing Factor-Independent Parthanatos Cell Death via Promoting PARP-1 Hyperactivation and Mitochondrial Dysfunction.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a scaffold protein and plays critical roles in neuronal synaptic formation and brain development. Previously, CASK was shown to associate with EGFR to maintain the vulval cell differentiation in C. elegans. In this study, we explored the role of CASK in CHME3 microglial cells. We found that CASK silencing protects cells from H2O2-induced cell death by attenuating PARP-1 activation, mitochondrial membrane potential loss, reactive oxygen species production, and mitochondrial fission, but it increases oxidative phosphorylation. The PARP-1 inhibitor olaparib blocks H2O2-induced cell death, suggesting the death mode of parthanatos. CASK silencing also increases AKT activation but decreases AMPK activation under H2O2 treatment. Pharmacological data further indicate that both signaling changes contribute to cell protection. Different from the canonical parthanatos pathway, we did not observe the AIF translocation from mitochondria into the nucleus, suggesting a non-canonical AIF-independent parthanatos in H2O2-treated CHME3 cells. Moreover, we found that CASK silencing upregulates the EGFR gene and protein expression and increases H2O2-induced EGFR phosphorylation in CHME3 microglia. However, EGFR activation does not contribute to cell protection caused by CASK silencing. In conclusion, CASK plays a crucial role in microglial parthanatos upon H2O2 treatment via stimulation of PARP-1 and AMPK but the inhibition of AKT. These findings suggest that CASK might be an ideal therapeutic target for CNS disorders.

Brexpiprazole suppresses cell proliferation and de novo lipogenesis through AMPK/SREBP1 pathway in colorectal cancer.

In the present study, we investigated the role of brexpiprazole on cell proliferation and lipogenesis in colorectal cancer (CRC) and its molecular mechanism. The effect of brexpiprazole on CRC cell proliferation was determined by CCK-8, EdU assay, cell clone formation. The flow cytometry was evaluated cell cycle. Differential expression genes (DEGs) were identified by RNA-seq assay after treating HCT116 cells with or without 20 μM brexpiprazole for 24 h. Then, the top 120 DEGs were analyzed by GO and KEGG enrichment analysis. After that, Oil red O staining and the levels of total cholestenone and triglyceride were measured to assess lipogenesis capacity in CRC cells. The related molecules of cell proliferation, lipogenic and AMPK/SREBP1 signal pathways were measured by q-PCR, western blot and immunohistochemical staining. Brexpiprazole remarkably suppressed cell proliferation, lipogenesis, and induced cell cycle arrest in CRC. The underlying mechanisms probably involved the suppression of SREBP1 and the stimulation of AMPK. Brexpiprazole inhibited cell proliferation and de novo lipogenesis through AMPK/SREBP1 pathway in CRC.

Mapping the metabolic reprogramming induced by sodium-glucose cotransporter 2 inhibition.

Diabetes is associated with increased risk for kidney disease, heart failure, and mortality. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) prevent these adverse outcomes; however, the mechanisms involved are not clear. We generated a roadmap of the metabolic alterations that occur in different organs in diabetes and in response to SGLT2i. In vivo metabolic labeling with 13C-glucose in normoglycemic and diabetic mice treated with or without dapagliflozin, followed by metabolomics and metabolic flux analyses, showed that, in diabetes, glycolysis and glucose oxidation are impaired in the kidney, liver, and heart. Treatment with dapagliflozin failed to rescue glycolysis. SGLT2 inhibition increased glucose oxidation in all organs; in the kidney, this was associated with modulation of the redox state. Diabetes was associated with altered methionine cycle metabolism, evident by decreased betaine and methionine levels, whereas treatment with SGLT2i increased hepatic betaine along with decreased homocysteine levels. mTORC1 activity was inhibited by SGLT2i along with stimulation of AMPK in both normoglycemic and diabetic animals, possibly explaining the protective effects against kidney, liver, and heart diseases. Collectively, our findings suggest that SGLT2i induces metabolic reprogramming orchestrated by AMPK-mTORC1 signaling with common and distinct effects in various tissues, with implications for diabetes and aging.

Adiponectin receptor agonist AdipoRon improves skeletal muscle function in aged mice.

The loss of skeletal muscle function with age, known as sarcopenia, significantly reduces independence and quality of life and can have significant metabolic consequences. Although exercise is effective in treating sarcopenia it is not always a viable option clinically, and currently, there are no pharmacological therapeutic interventions for sarcopenia. Here, we show that chronic treatment with pan-adiponectin receptor agonist AdipoRon improved muscle function in male mice by a mechanism linked to skeletal muscle metabolism and tissue remodeling. In aged mice, 6 weeks of AdipoRon treatment improved skeletal muscle functional measures in vivo and ex vivo. Improvements were linked to changes in fiber type, including an enrichment of oxidative fibers, and an increase in mitochondrial activity. In young mice, 6 weeks of AdipoRon treatment improved contractile force and activated the energy-sensing kinase AMPK and the mitochondrial regulator PGC-1a (peroxisome proliferator-activated receptor gamma coactivator one alpha). In cultured cells, the AdipoRon induced stimulation of AMPK and PGC-1a was associated with increased mitochondrial membrane potential, reorganization of mitochondrial architecture, increased respiration, and increased ATP production. Furthermore, the ability of AdipoRon to stimulate AMPK and PGC1a was conserved in nonhuman primate cultured cells. These data show that AdipoRon is an effective agent for the prevention of sarcopenia in mice and indicate that its effects translate to primates, suggesting it may also be a suitable therapeutic for sarcopenia in clinical application.

Stimulation of AMPK Prevents Diabetes-Induced Photoreceptor Cell Degeneration.

Diabetic retinopathy (DR) is a kind of severe retinal neurodegeneration. The advanced glycation end products (AGEs) affect autophagy, and mitochondrial function is involved in DR. Adenosine-activated protein kinase (AMPK) is an important metabolic sensor that can regulate energy homeostasis in cells. However, the effect of AMPK in DR is still not fully understood. In this study, we investigated the effect of AMPK on diabetes-induced photoreceptor cell degeneration. In vivo, a diabetic mouse model was established by streptozotocin (STZ) injection. Haematoxylin-eosin (HE) staining was used to observe retinal morphology and measure the thicknesses of different layers in the retina. Electroretinogram (ERG) was used to evaluate retinal function. In vitro, 661w cells were treated with AGEs with/without an AMPK agonist (metformin) or AMPK inhibitor (compound C). Flow cytometry and CCK-8 assays were used to analyse apoptosis. Mitochondrial membrane potential was analysed by JC-1. Western blotting and qRT-PCR were used to examine the expression of related proteins and genes, respectively. The wave amplitude and the thickness of the outer nuclear layer were decreased in diabetic mice. The expression of rhodopsin and opsin was also decreased in diabetic mice. In vitro, the percentage of apoptotic cells was increased, the expression of the apoptosis-related protein Bax was increased, and Bcl-2 was decreased after AGE treatment in 661w cells. The expression of the autophagy-related protein LC3 was decreased, and p62 was increased. The mitochondrial-related gene expression and membrane potential were decreased, and mitochondrial morphology was abnormal, as observed by TEM. However, AMPK stimulation ameliorated this effect. These results indicate that AMPK stimulation can delay diabetes-induced photoreceptor degeneration by regulating autophagy and mitochondrial function.

Mechanisms by which adiponectin reverses high fat diet-induced insulin resistance in mice

Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains unclear. Two weeks of globular adiponectin (gAcrp30) treatment reduced fasting plasma glucose, triglyceride (TAG), and insulin concentrations and reversed whole-body insulin resistance, which could be attributed to both improved insulin-mediated suppression of endogenous glucose production and increased insulin-stimulated glucose uptake in muscle and adipose tissues. These improvements in liver and muscle sensitivity were associated with ∼50% reductions in liver and muscle TAG and plasma membrane (PM)-associated diacylglycerol (DAG) content and occurred independent of reductions in total ceramide content. Reductions of PM DAG content in liver and skeletal muscle were associated with reduced PKCε translocation in liver and reduced PKCθ and PKCε translocation in skeletal muscle resulting in increased insulin-stimulated insulin receptor tyrosine1162 phosphorylation, IRS-1/IRS-2-associated PI3-kinase activity, and Akt-serine phosphorylation. Both gAcrp30 and full-length adiponectin (Acrp30) treatment increased eNOS/AMPK activation in muscle and muscle fatty acid oxidation. gAcrp30 and Acrp30 infusions also increased TAG uptake in epididymal white adipose tissue (eWAT), which could be attributed to increased lipoprotein lipase (LPL) activity. These data suggest that adiponectin and adiponectin-related molecules reverse lipid-induced liver and muscle insulin resistance by reducing ectopic lipid storage in these organs, resulting in decreased plasma membrane sn-1,2-DAG-induced nPKC activity and increased insulin signaling. Adiponectin mediates these effects by both promoting the storage of TAG in eWAT likely through stimulation of LPL as well as by stimulation of AMPK in muscle resulting in increased muscle fat oxidation.

A Phase 2 Study of Neo-adjuvant Metformin and Temozolomide followed by Hypofractionated Accelerated RadioTherapy (HART) with Concomitant and Adjuvant Metformin and Temozolomide (TMZ) in Patients with Glioblastoma

Metformin inhibits the proliferation of glioblastoma (GBM) cancer stem cells through stimulation of AMPK, lowers glucose levels, insulin, and IGF-1. The purpose was to assess the feasibility of post-op neo-adjuvant metformin and TMZ, followed by HART with concomitant and adjuvant TMZ/metformin. Neo-adjuvant metformin (850 mg BID) and TMZ (75 mg/m2 QD) were started within 4 weeks from surgery, for 14 days prior to starting radiotherapy. HART was delivered using IMRT simultaneous boost technique. The CTV included surgical resection cavity and post-operative residual contrast enhancing lesion. The PTV_60 included CTV+3-5 mm margin, while PTV_40 included CTV + 15 mm. A dose of 60 Gy was delivered to PTV_60 while a dose of 40 Gy was delivered to PTV_40 in 20 daily fractions. Concomitant and adjuvant TMZ were administered as per the Stupp’s schedule with concomitant metformin. Thirty-three patients, 19 females and 14 males were included. Median age was 54 years, median ECOG score was 1 At a median follow-up of 20.3 months (range 2.0-55.8) for patients at risk, the median survival was 38.2 months (95% CI, 12-59), with 20 out of 33 patients alive at the date of this report. At 2 years the overall survival was 65.5%, and 50.5 % at 3 years. Median PFS was 15.4 months (95% CI, 10-36). For 27 patients with gross total resection, median survival was 38.84 months compared to 16.8 months for 6 patients with partial resection (p = 0.02). The median survival of patients aged < 60 years was 38.8 months and 17.1 for patients > 60 years (p = 0.16). The median survival for 12 patients with methylated MGMT was not reached compared to 38.2 months for 21 patients with unmethylated MGMT (p = 0.12). Adjuvant TMZ/Metformin was discontinued in 2 patients, one with disease progression, and another one following prolonged grade 4 thrombocytopenia. There were no unexpected severe adverse events. Thirteen patients were re-operated for findings suggestive of disease progression. Six patients of the 13 re-operated patients had treatment-related necrosis with no viable residual GBM. Two of these 6 patients later developed disease progression. Eighty-one percent and 70.9% of the patients were free from treatment-related necrosis at 2 and 3 years, respectively. Neo-adjuvant metformin and TMZ followed by concurrent TMZ/metformin and HART and adjuvant TMZ/metformin is feasible and safe, and the results compare favorably to current GBM literature, especially for patients with unmethylated MGMT.

The role of AMP-activated protein kinase in the expression of the dystrophin-associated protein complex in skeletal muscle.

Stimulation of AMPK induces the expression of dystrophin-associated protein complex (DAPC) components in skeletal muscle, whereas reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast, glycolytic extensor digitorum longus (EDL) and slow, oxidative soleus (Sol) muscles from wild-type mice and from littermates with skeletal muscle-specific knockout of the AMPK β1 and β2 subunits (AMPK β1 β2M-KO; MKO) were analyzed. DAPC mRNA and protein expression were similar between genotypes, with the exception of elevated neuronal nitric oxide synthase expression at the sarcolemma in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC was also not affected by the loss of AMPK. However, MyoD and myogenin expression was diminished in MKO muscles, consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO Sol muscles, likely due to the adaptive accumulation of peroxisome proliferator-activated receptor γ coactivator-1α at the sarcolemma of MKO EDL muscles. Collectively, the evidence indicates that AMPK is sufficient but not essential for the maintenance of DAPC expression in skeletal muscle, yet it is required for preserving extrasynaptic utrophin levels in slow oxidative muscles.-Dial, A. G., Rooprai, P., Lally, J. S., Bujak, A. L., Steinberg, G. R., Ljubicic, V. The role of AMP-activated protein kinase in the expression of the dystrophin-associated protein complex in skeletal muscle.

Effect of A-769662, a direct AMPK activator, on Tlr-4 expression and activity in mice heart tissue.

Objective(s)::TLR-4 activates a number of inflammatory signaling pathways. Also, AMPK could be involved in anti-inflammatory signaling. The aim of this study was to identify whether stimulation of AMPK could inhibit LPS-induced Tlr-4 gene expression in mice hearts.Materials and methods:Heart AMPK activity and/or Tlr-4 expression was stimulated in different mice groups, using respectively IP injection of A-769662 (10 mg/kg) and LPS (2 mg/kg) or a combination of both agents. Moreover, compound-C (20 mg/kg), as an AMPK antagonist, was intraperitoneally co-administrated with both A-769662 and LPS in another group to investigate the role of AMPK activity on Tlr-4 regulation. After 8 hr, in addition to peripheral neutrophil cell count, myocardial p-AMPK, p-ACC as well as MyD88 protein contents and Tlr-4 expression was assessed by Western blotting and real-time qRT-PCR, respectively. TNF-α and IL-6 expression levels were also determined by ELISA.Results:LPS induced heart Tlr-4 expression (P<0.001) associating with an increase in the myocardial MyD88 protein content (P<0.001), elevation of heart TNF-α (P<0.01) and IL-6 (P<0.05) concentrations, and rise in the peripheral neutrophil cell count (P<0.001). Administration of A-769662 decreased LPS-induced Tlr-4 expression (P<0.01) and alleviated peripheral neutrophil cell count (P<0.01). The inhibitory effect of A-769662 on LPS-induced Tlr-4 expression was reversed by antagonizing AMPK with compound-C (P<0.001) which reduced p-AMPK (P<0.05) and p-ACC (P<0.01) myocardial protein contents in the LPS+A-769662 group.Conclusion:This study demonstrated that activation of AMPK, by A-769662 agent, could inhibit Tlr-4 expression and activity, suggesting a link between AMPK and Tlr-4 in heart tissue.

High-molecular-weight cocoa procyanidins possess enhanced insulin-enhancing and insulin mimetic activities in human primary skeletal muscle cells compared to smaller procyanidins

Dysregulation of glucose metabolism is a primary hallmark of metabolic disease (i.e., diabetes, obesity, etc.). Complementary nonpharmaceutical strategies are needed to prevent and/or ameliorate dysregulation of glucose metabolism and prevent progression from normoglycemia to prediabetes and type 2 diabetes across the lifespan. Cocoa compounds, particularly the procyanidins, have shown promise for improving insulin sensitivity and blood glucose homeostasis. However, the molecular mechanisms by which cocoa procyanidins exert these functions remain poorly understood. Furthermore, cocoa procyanidins exhibit size diversity, and evidence suggests that procyanidin bioactivity and size may be related. Here, we show that a procyanidin-rich cocoa extract elicits an antidiabetic effect by stimulating glycogen synthesis and glucose uptake, independent of insulin. Cocoa procyanidins did not appear to act via stimulation of AMPK or CaMKII activities. Additionally, in the presence of insulin, glycogen synthesis and AKT phosphorylation were affected. These mechanisms of action are most pronounced in response to oligomeric and polymeric procyanidins. These results demonstrate (1) specific mechanisms by which cocoa procyanidins improve glucose utilization in skeletal muscle and (2) that larger procyanidins appear to possess enhanced activities. These mechanistic insights suggest specific strategies and biological contexts that may be exploited to maximize the antidiabetic benefits of cocoa procyanidins.

Hypoxia upregulates Malat1 expression through a CaMKK/AMPK/HIF-1α axis.

Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21%O2 conditions, HeLa cells incubated in 1.5%O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21%O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21%O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway.

Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells

High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.

Contraction-stimulated glucose transport in muscle is controlled by AMPK and mechanical stress but not sarcoplasmatic reticulum Ca(2+) release.

Understanding how muscle contraction orchestrates insulin-independent muscle glucose transport may enable development of hyperglycemia-treating drugs. The prevailing concept implicates Ca2+ as a key feed forward regulator of glucose transport with secondary fine-tuning by metabolic feedback signals through proteins such as AMPK. Here, we demonstrate in incubated mouse muscle that Ca2+ release is neither sufficient nor strictly necessary to increase glucose transport. Rather, the glucose transport response is associated with metabolic feedback signals through AMPK, and mechanical stress-activated signals. Furthermore, artificial stimulation of AMPK combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response. These results suggest that ATP-turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction, and call for a major reconsideration of the established Ca2+ centric paradigm.

The Role of Macrophage Migration Inhibitory Factor in Anesthetic-Induced Myocardial Preconditioning

IntroductionAnesthetic-induced preconditioning (AIP) is known to elicit cardioprotective effects that are mediated at least in part by activation of the kinases AMPK and PKCε as well as by inhibition of JNK. Recent data demonstrated that the pleiotropic cytokine macrophage migration inhibitory factor (MIF) provides cardioprotection through activation and/or inhibition of kinases that are also known to mediate effects of AIP. Therefore, we hypothesized that MIF could play a key role in the AIP response.MethodsCardiomyocytes were isolated from rats and subjected to isoflurane preconditioning (4 h; 1.5 vol. %). Subsequently, MIF secretion and alterations in the activation levels of protective kinases were compared to a control group that was exposed to ambient air conditions. MIF secretion was quantified by ELISA and AIP-induced activation of protein kinases was assessed by Western blotting of cardiomyocyte lysates after isoflurane treatment.ResultsIn cardiomyocytes, preconditioning with isoflurane resulted in a significantly elevated secretion of MIF that followed a biphasic behavior (30 min vs. baseline: p = 0.020; 24 h vs. baseline p = 0.000). Moreover, quantitative polymerase chain reaction demonstrated a significant increase in MIF mRNA expression 8 h after AIP. Of note, activation of AMPK and PKCε coincided with the observed peaks in MIF secretion and differed significantly from baseline.ConclusionsThese results suggest that the pleiotropic mediator MIF is involved in anesthetic-induced preconditioning of cardiomyocytes through stimulation of the protective kinases AMPK and PKCε.

Adiponectin Mediated APPL1-AMPK Signaling Induces Cell Migration, MMP Activation, and Collagen Remodeling in Cardiac Fibroblasts

Defects in adiponectin action have been implicated in the development of cardiac dysfunction in obesity and diabetes. Cardiac fibroblasts play an important role in regulating extracellular matrix remodeling yet little is known regarding the direct effects of adiponectin on cardiac fibroblasts. In this study, we first demonstrated temporal relocalization of cellular APPL1 in response to adiponectin in primary cardiac fibroblasts and that siRNA-mediated knockdown of APPL1 attenuated stimulation of AMPK by adiponectin. The cell surface content of MT1-MMP and activation of MMP2 were induced by adiponectin and these responses were dependent on AMPK signaling. Enhanced MMP activity facilitated increased fibroblast migration in response to adiponectin which was also prevented by inhibition of AMPK, with no change in cell proliferation observed. Collagen and elastin immunofluorescence demonstrated reorganization of the extracellular matrix in accordance with increased MMP activity, whereas quantitative mRNA analysis, (3) H-proline incorporation and picrosirius red assays showed no change in intracellular or extracellular total collagen levels in response to adiponectin. In summary, these data are the first to report the adiponectin stimulated APPL1-AMPK signaling axis in cardiac fibroblasts and characterize MT1-MMP translocation, MMP2 activity and cell migration as functional outcomes. These effects may be of significance in heart failure associated with obesity and diabetes.

Resveratrol Induces Vascular Smooth Muscle Cell Differentiation through Stimulation of SirT1 and AMPK

Phenotypic plasticity in vascular smooth muscle cells (VSMC) is necessary for vessel maintenance, repair and adaptation to vascular changes associated with aging. De-differentiated VSMC contribute to pathologies including atherosclerosis and intimal hyperplasia. As resveratrol has been reported to have cardio- protective effects, we investigated its role in VSMC phenotypic modulation. We demonstrated the novel finding that resveratrol promoted VSMC differentiation as measured by contractile protein expression, contractile morphology and contraction in collagen gels. Resveratrol induced VSMC differentiation through stimulation of SirT1 and AMPK. We made the novel finding that low or high dose resveratrol had an initially different mechanism on induction of differentiation. We found that low dose resveratrol stimulated differentiation through SirT1-mediated activation of AKT, whereas high dose resveratrol stimulated differentiation through AMPK-mediated inhibition of the mTORC1 pathway, allowing activation of AKT. The health effects of resveratrol in cardiovascular diseases, cancer and longevity are an area of active research. We have demonstrated a supplemental avenue where-by resveratrol may promote health by maintaining and enhancing plasticity of the vasculature.

Synergistic effects of polyphenols with leucine and β‐hydroxy‐ β‐methylbutyrate (HMB) on energy metabolism

We recently demonstrated synergy between low dose resveratrol (200 nM) with leucine or its metabolite HMB on energy metabolism. We have now assessed potential synergy of other polyphenols with leucine and HMB. Dose‐response curves for fatty acid oxidation (FAO) were constructed and the highest non‐effective dose (typically 1 – 10 nM) was combined with either leucine (0.5 mM) or HMB (5 μM) to treat adipocytes and myotubes for 24h. Significant synergy was detected for the stilbenes (resveratrol, piceatannol) and a stilbene‐containing grape seed extract, resulting in stimulation of FAO in adipocytes (60–70%, p&lt;0.05) and myotubes (&gt;;2000%, p&lt;0.01) and stimulation of AMPK (~50%, p&lt;0.03) and Sirt1 (~65%, p&lt;0.001) activities. Hydroxycinnamic acids, including chlorogenic and cinnamic acids and their derivatives, exhibited robust synergy with leucine and HMB, increasing FAO (300–1300% p&lt;0.01), AMPK activity (~50%–150%, p&lt;0.01), Sirt1 activity (~70%, p&lt;0.001) and insulin sensitivity/glucose utilization (~50 – 150%, p&lt;0.05). In contrast, more complex polyphenol structures (ellagic acid, epigallocatechin gallate) required higher concentrations (&gt;;1 μM) and exerted little or no synergy with leucine or HMB. These data indicate synergy of several naturally occurring polyphenols with leucine or HMB in concentrations which produce no independent effects and are readily achievable via dietary supplementation.

Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H2O2) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H2O2-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H2O2 (100μM) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5min (Δ 183%, P<0.05), 3min (Δ 414%, P<0.05), and 10min (Δ 35%, P<0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H2O2>. Furthermore, H2O2 inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10min; Δ −32%, P<0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPKα; 78% at 3min and 244% at 10min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100μM, 2h). Moreover, Compound C significantly reduced the effect of H2O2 on IR phosphorylation by about 40% (from 2.07±0.28 to 1.28±0.12, P<0.05). In addition, H2O2 increased glucose uptake in podocytes (from 0.88±0.04 to 1.29±0.12nmol/min/mg protein, P<0.05), and this effect was attenuated by Compound C.Our results suggested that H2O2 activated the insulin signaling pathway and glucose uptake via AMPK in cultured rat podocytes. This signaling may play a potential role in the prevention of insulin resistance under conditions associated with oxidative stress.

Stimulation or Inhibition of AMPK Causes Arrest of Mouse Embryo Development.

Stimulation or Inhibition of AMPK Causes Arrest of Mouse Embryo Development.

Metformin Inhibits Aromatase Expression in Primary Human Breast Adipose Stromal Cells.

Abstract There is good epidemiological evidence that obesity is linked to an increased risk of cancers such as colon and breast. Given the obesity problem worldwide, it is now believed that tens of millions more women may contract breast cancer in their senior years than was previously thought. After menopause, when the ovaries have ceased to produce estrogens, it is the local expression of aromatase, the enzyme responsible for converting androgens to estrogens, within the breast adipose that is believed to be responsible for the increased proliferation of breast cancer cells.We have recently demonstrated that the LKB1/AMPK pathway is inhibitory of aromatase expression in primary human breast adipose stromal cells by inhibiting the nuclear entry of CRTC2, a CREB co-activator. Factors produced in obesity, such as leptin, and by tumors, such as PGE2, were shown to inhibit LKB1 expression and activity and cause the nuclear translocation of CRTC2, resulting in the increased expression of aromatase. Conversely, a factor produced in lean individuals, adiponectin, and the drug AICAR, known to activate AMPK, inhibited the PGE2-mediated expression of aromatase.There is growing evidence that the action of metformin, an oral anti-diabetic drug which has been used for many years, is mediated primarily by stimulation of AMPK, in association with interaction with its upstream kinase LKB1. In the present study, we aimed to examine the effect of metformin on aromatase expression and on the LKB1/AMPK pathway within the breast. Primary human breast adipose stromal cells obtained from breast reduction surgeries were treated with increasing amounts of 1,1-dimethylbiguanide hydrochloride (metformin) and aromatase transcript expression was quantified using real-time PCR. Interestingly, 24 hour treatment with metformin resulted in the dose-dependent inhibition of aromatase expression at concentrations of 10 and 50 uM. LKB1 activity was measured by examining the phosphorylation of AMPK by Western blotting and consistent with the proposed role of LKB1/AMPK in aromatase expression, metformin caused a significant increase in AMPK phosphorylation.Phase III aromatase inhibitors are proving highly successful as endocrine therapy for breast cancer in post-menopausal women, replacing Tamoxifen as frontline, second line and neoadjuvant therapy. However long-term contraindications such as bone loss, arthralgia, adverse cardiovascular effects and possibly cognitive defects may limit the use of these compounds in the prevention setting. Moreover, aromatase expression may be only one of several pathways whereby obesity influences breast cancer development. This study suggests that using a drug such as metformin which increases insulin sensitivity, causes weight loss and decreases the local biosynthesis of estrogens, may provide a multi-facetted approach at preventing and treating obesity-related postmenopausal estrogen-dependent breast cancer.Work supported by NHMRC project grant 494819 and the Terry Fox Foundation Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3132.