Increase In AMPK Phosphorylation Research Articles

A novel blood-based bioassay to monitor adiponectin signaling

The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-β release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.

Homer1 Protects against Retinal Ganglion Cell Pyroptosis by Inhibiting Endoplasmic Reticulum Stress-Associated TXNIP/NLRP3 Inflammasome Activation after Middle Cerebral Artery Occlusion-Induced Retinal Ischemia.

Retinal ischemia, after cerebral ischemia, is an easily overlooked pathophysiological problem in which inflammation is considered to play an important role. Pyroptosis is a kind of cell death pattern accompanied by inflammation. Homer scaffold protein 1 (Homer1) has anti-inflammation properties and protects against ischemic injury. However, little is known about pyroptosis following middle cerebral artery occlusion (MCAO)-induced retinal ischemia and the regulatory mechanisms involved by Homer1 for the development of pyroptosis. In the present study, retinal ischemic injury was induced in mice by permanent MCAO in vivo, and retinal ganglion cells (RGCs) were subjected to Oxygen and Glucose Deprivation (OGD) to establish an in vitro model. It was shown that TXNIP/NLRP3-mediated pyroptosis was located predominantly in RGCs, which gradually increased after retinal ischemia and peaked at 24 h after retinal ischemia. Interestingly, the RGCs pyroptosis occurred not only in the cell body but also in the axon. Notably, the occurrence of pyroptosis coincided with the change of Homer1 expression in the retina after retinal ischemia and Homer1 also co-localized with RGCs. It was demonstrated that overexpression of Homer1 not only alleviated RGCs pyroptosis and inhibited the release of pro-inflammatory factors but also led to the increase in phosphorylation of AMPK, inhibition of ER stress, and preservation of visual function after retinal ischemia. In conclusion, it was suggested that Homer1 may protect against MCAO-induced retinal ischemia and RGCs pyroptosis by inhibiting endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation after MCAO-induced retinal ischemia.

Metformin suppresses SARS-CoV-2 in cell culture

Comorbidities such as diabetes worsen COVID-19 severity and recovery. Metformin, a first-line medication for type 2 diabetes, has antiviral properties and certain studies have also indicated its prognostic potential in COVID-19. Here, we report that metformin significantly inhibits SARS-CoV-2 growth in cell culture models. First, a steady increase in AMPK phosphorylation was detected as infection progressed, suggesting its important role during viral infection. Activation of AMPK in Calu3 and Caco2 cell lines using metformin revealed that metformin suppresses SARS-CoV-2 infectious titers up to 99%, in both naïve as well as infected cells. IC50 values from dose-variation studies in infected cells were found to be 0.4 and 1.43 mM in Calu3 and Caco2 cells, respectively. Role of AMPK in metformin's antiviral suppression was further confirmed using other pharmacological compounds, AICAR and Compound C. Collectively, our study demonstrates that metformin is effective in limiting the replication of SARS-CoV-2 in cell culture and thus possibly could offer double benefits as diabetic COVID-19 patients by lowering both blood glucose levels and viral load.

PIM3-AMPK-HDAC4/5 axis restricts MuERVL-marked 2-cell-like state in embryonic stem cells.

SummaryA minority of embryonic stem cells (ESCs) marked by endogenous retrovirus MuERVL are totipotent 2-cell-like cells. However, the majority of ESCs repress MuERVL. Currently, it is still unclear regarding the signaling pathway(s) repressing the MuERVL-associated 2-cell-like state of ESCs. Here, we identify the PIM3-downstream signaling axis as a key route to repress MuERVL and 2-cell-like state. Downregulation, deletion, or inhibition of PIM3 activated MuERVL, 2-cell genes, and trophectodermal genes in ESCs. By screening PIM3-regulated pathways, we discovered AMPK as its key target. The loss of Pim3 caused an increase in AMPK phosphorylation, which phosphorylated HDAC4/5 and triggered their transfer out of the nucleus in Pim3−/− ESCs. The reduction of nuclear HDAC4/5 caused increased H3K9ac and reduced H3K9me1/2 enrichment on MuERVL, thus activating MuERVL and 2-cell-like state. In summary, our study uncovers a novel axis by which PIM3 suppresses 2-cell marker MuERVL and totipotent state in ESCs.

Social isolation triggers oxidative status and impairs systemic and hepatic insulin sensitivity in normoglycemic rats

Drug-naïve psychotic patients show metabolic and hepatic dysfunctions. The rat social isolation model of psychosis allows to investigate mechanisms leading to these disturbances to which oxidative stress crucially contributes. Here, we investigated isolation-induced central and peripheral dysfunctions in glucose homeostasis and insulin sensitivity, along with redox dysregulation. Social isolation did not affect basal glycemic levels and the response to glucose and insulin loads in the glucose and insulin tolerance tests. However, HOMA-Index value were increased in isolated (ISO) rats. A hypothalamic reduction of AKT phosphorylation and a trend toward an increase in AMPK phosphorylation were observed following social isolation, accompanied by reduced GLUT-4 levels. Social isolation also induced a reduction of phosphorylation of the insulin receptor, of AKT and GLUT-2, and a decreased phosphorylation of AMPK in the liver. Furthermore, a significant reduction in hepatic CPT1 and PPAR-α levels was detected. ISO rats also showed significant elevations in hepatic ROS amount, lipid peroxidation and NOX4 expression, whereas no differences were detected in NOX2 and NOX1 levels. Expression of SOD2 in the mitochondrial fraction and SOD1 in the cytosolic fraction was not altered following social isolation, whereas SOD activity was increased. Furthermore, a decrease of hepatic CAT and GSH amount was observed in ISO rats compared to GRP animals. Our data suggest that the increased oxidant status and antioxidant capacity modifications may trigger hepatic and systemic insulin resistance, by altering signal hormone pathway and sustaining subsequent alteration of glucose homeostasis and metabolic impairment observed in the social isolation model of psychosis.

MyD88‐mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms

Skeletal muscle atrophy is a debilitating complication of many chronic disease states and disuse conditions including denervation. However, molecular and signaling mechanisms of muscle wasting remain less understood. Here, we demonstrate that the levels of several toll-like receptors (TLRs) and their downstream signaling adaptor, myeloid differentiation primary response 88 (MyD88), are induced in skeletal muscle of mice in response to sciatic nerve denervation. Muscle-specific ablation of MyD88 mitigates denervation-induced skeletal muscle atrophy in mice. Targeted ablation of MyD88 suppresses the components of ubiquitin-proteasome system, autophagy, and FOXO transcription factors in skeletal muscle during denervation. We also found that specific inhibition of MyD88 reduces the activation of canonical nuclear factor-kappa (NF-κB) pathway and expression of receptors for inflammatory cytokines in denervated muscle. In contrast, inhibition of MyD88 stimulates the activation of non-canonical NF-κB signaling in denervated skeletal muscle. Ablation of MyD88 also inhibits the denervation-induced increase in phosphorylation of AMPK without having any effect on the phosphorylation of mTOR. Moreover, targeted ablation of MyD88 inhibits the activation of a few components of the unfolded protein response (UPR) pathways, especially X-box protein 1 (XBP1). Importantly, myofiber-specific ablation of XBP1 mitigates denervation-induced skeletal muscle atrophy in mice. Collectively, our experiments suggest that TLR-MyD88 signaling mediates skeletal muscle wasting during denervation potentially through the activation of canonical NF-κB signaling, AMPK and UPR pathways.

Asparagus officinalis Exhibits Anti-Tumorigenic and Anti-Metastatic Effects in Ovarian Cancer.

Ovarian cancer is one of the leading causes of female cancer death. Emerging evidence suggests that many dietary natural products have anti-tumorigenic activity, including that of asparagus officinalis. The current study aimed to assess the anti-tumorigenic and anti-metastatic effects of asparagus officinalis on serous ovarian cancer cell lines and a transgenic mouse model of high grade serous ovarian cancer. Asparagus officinalis decreased cellular viability, caused cell cycle G1 phase arrest and induced apoptosis in the OVCAR5 and SKOV3 cells. Induction of apoptosis and inhibition of cell proliferation was rescued by the pan-caspase inhibitor, Z-VAD-FMK, implying that its cytotoxic effects were mainly dependent on caspase pathways. Asparagus officinalis increased levels of ROS and decreased mitochondrial membrane potential with corresponding increases in PERK, Bip, Calnexin PDI and ATF4 in both cell lines. Treatment with asparagus officinalis also reduced ability of adhesion and invasion through epithelial–mesenchymal transition and reduction of VEGF expression. The combination of Asparagus officinalis with paclitaxel had synergistic anti-proliferative activity. Furthermore, Asparagus officinalis significantly inhibited tumor growth and reduced serum VEGF in a genetically engineered mouse model of ovarian cancer under obese and lean conditions, accompanied with a decrease in the expression of Ki67, VEGF and phosphorylated S6, and in an increase in phosphorylation of AMPK in the ovarian tumor tissues. Overall, our data provide a pre-clinical rationale for asparagus officinalis in the prevention and treatment of ovarian cancer as a novel natural product.

Selinexor in Combination with Induction and Consolidation Therapy in Older Adults with AML Is Highly Active

Background: Acute myeloid leukemia (AML) is an aggressive myeloid cancer of the hematopoietic system that primarily affects older adults and is characterized by therapy resistance and dismal outcomes. Novel approaches to treat AML are desperately needed. Selinexor is a small-molecule inhibitor of the nuclear export protein XPO1. Treatment of AML cells with selinexor was shown to sensitize them to chemotherapy in part by blocking the nuclear export of topoisomerase II, resulting in increased DNA strand breaks when an anthracycline is present. Furthermore, selinexor has shown encouraging results when combined with chemotherapy in the relapsed setting. This abstract reports on the initial results of a randomized phase II study of induction and consolidation with or without selinexor in newly diagnosed patients with AML 60 years of age or older. Methods: Patients 60 years of age or older with newly diagnosed de novo AML were randomized between 7+3 or 7+3+selinexor induction. Responding patients could then go on to high dose cytarabine consolidation with or without selinexor as per their initial treatment assignment. Patients in the selinexor arm who completed all planned consolidation could then move to maintenance therapy with selinexor alone. During induction cytarabine was dosed at 100 mg/m2 by continuous infusion for 7 days and daunorubicin was dosed at 60 mg/m2 on days 1-3. In consolidation cytarabine was dosed at 1.5 gm/m2 given Q12 hours on days 1, 3 and 5. Selinexor was dosed at 60 mg PO on days 1, 3, 8, 10, 15 and 17 during induction and consolidation and on days 1 and 8 every 21 days in maintenance. Optional blood samples were collected just prior to and at several time points after selinexor during induction. Mononuclear cells were isolated from these samples, lysed and lysates evaluated for protein pathway drug target activation mapping by reverse phase phosphoprotein array (RPPA). Results: Thirteen patients have been enrolled to date with 12 available for evaluation. Of the 12 evaluable patients 6 were randomized to each arm. Baseline demographics are listed in Table 1. Overall the combination was well tolerated with no patients discontinuing selinexor secondary to treatment related adverse events. In the standard arm 3 of 6 patients achieved a complete remission. Of the 3 responders 1 has relapsed, 1 has gone on to transplant and one has completed consolidation chemotherapy. In the selinexor arm 5 of 6 patients achieved a complete remission and 1 patient achieved a morphologic leukemia free state. All patients given selinexor achieved a response. Of the 5 responders 3 have gone on to transplant, 1 is on maintenance therapy (completed 14 cycles) and one is completing consolidation therapy. In the standard arm no patients died during induction and 3 of 6 patients have died from progressive disease. In the selinexor arm 1 patient died during induction and none of the remaining patients have progressed (Figure 1 and Table 2). No difference in the AE profile was noted between arms and no unexpected side effects were observed. The patient on long term maintenance selinexor is tolerating it without significant AEs to date. Blood samples were available from 3 patients from the selinexor arm. RPPA based protein pathway activation mapping analysis of PBMCs revealed a significant increase in phosphorylation of AMPK, BAD and LC3B at one-hour post treatment that returned to baseline by 2 hours suggesting a transient increase in these pathways that was subsequently suppressed (Figure 2). Conclusions: Selinexor in combination with standard induction and consolidation therapy appears highly active in older de novo AML patients. Selinexor may increase response by modulation of AMPK, autophagy and BAD phosphorylation. Enrollment is ongoing. Disclosures Pardee: Spherix Intellectual Property: Research Funding; CBM Bipharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Celgene: Speakers Bureau; Pharmacyclics/Janssen: Speakers Bureau; Karyopharm: Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding. Manuel:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau. Powell:Pfizer: Consultancy, Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Janssen: Research Funding. OffLabel Disclosure: Selinexor is not approved for the treatment of AML

The SGLT2 Inhibitor Canagliflozin Induces Catabolic Transcriptional and Metabolic Switching and Increases FGF-21 in Mice with Dietary Obesity

Given the impact of SGLT2 inhibitors to reduce overall mortality and CV risk in T2D, improved understanding of cellular and transcriptional mechanisms responsible for metabolic effects is required. We fed C57BL/6J mice a 60% high fat diet (HFD) for 4 weeks prior to assignment to one of 3 groups: (1) 60% HFD, (2) HFD with canagliflozin (CANA, 25 mg/kg/day), and (3) HFD weight-matched (WM) to CANA via caloric restriction. Mice were analyzed after 4-8 weeks. CANA reduced fasting glucose by 47% (P<0.0001), improved glucose tolerance, and 25% lower body weight vs. HFD (p<0.0001). CANA promoted shift toward lipid utilization, with respiratory quotient <0.7 even during feeding, and a 3.7 and 2.0-fold increase in plasma and liver β-hydroxybutyrate (both p<0.01 vs. HFD). In parallel, liver glycogen and cholesterol were reduced by 55% and 28%, and triglycerides were shifted toward polyunsaturated species. To identify potential mechanisms responsible for these fasting-like phenotypes, we analyzed the molecular impact of CANA. Liver transcriptomic analysis revealed marked downregulation of lipogenesis, glycolysis, and steroid synthesis pathways (p<0.01), while the lipid oxidative regulatory genes PPARα and PGC1α were increased by 2.8 and 4-fold, respectively. CANA significantly increased hepatic FAD+ (2.4-fold) and AMP (1.3-fold), in parallel with a 12-fold increase in AMPK phosphorylation and 50% reduction in Tor-dependent phosphorylation of S6. Hepatic gene expression of FGF-21, a hormonal regulator of fatty acid oxidation and ketogenesis, was increased by 4.5-fold, with parallel 2.2-fold increase in plasma FGF-21 in CANA-treated mice (p<0.vs. HFD for both). These effects were independent of insulin sensitivity or glucagon signaling. Collectively, these data demonstrate CANA induction of a fasting-like state by coordinated transcriptional-metabolic reprogramming, and identify FGF-21 as a potential molecular metabolic switch. Disclosure S. Osataphan: None. C. Macchi: None. V. Sales: None. C. Kozuka: None. J.I. Chimene-Weiss: None. Y. Tangjaroenpaisan: None. J. Morningstar: None. R. Gerszten: None. M.E. Patti: Research Support; Self; Janssen Pharmaceuticals, Inc.. Other Relationship; Self; Xeris Pharmaceuticals, Inc.. Research Support; Self; Ethicon US, LLC., Coviden, MedImmune. Other Relationship; Self; Novo Nordisk Inc., XOMA Corporation, AstraZeneca, Nestlé. Research Support; Self; Dexcom, Inc.. Consultant; Self; Eiger BioPharmaceuticals.

Measures of Growth Processes and Myogenesis in Glycolytic and Oxidative Muscle Fibers in Rats after Indirect Electrical Stimulation

We report here a comparative study of activity in signal pathways and gene expression in the red (RGM) and white (WGM) parts of the gastrocnemius muscle in rats after series of short (1 sec) tetanic contractions evoked by stimulation of the motor nerve at a frequency of 100 Hz and an amplitude sufficient to activate all the motor units of the muscle. At 2 h after stimulation, WGM showed more marked increases in the level of ERK1/2 phosphorylation than RGM, though increases in AMPK phosphorylation were no different. Furthermore, the increases in MyoD and myogenin mRNA in WGM were significantly greater than those in RGM, while the effects of stimulation on expression of the IGF-1, MaFbx, and MuRF genes were weak and similar in WGM and RGM. There was also an increase in the content of myostatin mRNA in RGM. Thus, glycolytic muscle fibers in WGM display more marked regulatory hypertrophic-type shifts than the oxidative muscle fibers making up RGM.

Exercise duration-matched interval and continuous sprint cycling induce similar increases in AMPK phosphorylation, PGC-1α and VEGF mRNA expression in trained individuals.

PurposeThe effects of low-volume interval and continuous ‘all-out’ cycling, matched for total exercise duration, on mitochondrial and angiogenic cell signalling was investigated in trained individuals.MethodsIn a repeated measures design, 8 trained males (dot{V}{text{O}}_{{2{text{peak}}}}, 57 ± 7 ml kg−1 min−1) performed two cycling exercise protocols; interval (INT, 4 × 30 s maximal sprints interspersed by 4 min passive recovery) or continuous (CON, 2 min continuous maximal sprint). Muscle biopsies were obtained before, immediately after and 3 h post-exercise.ResultsTotal work was 53 % greater (P = 0.01) in INT compared to CON (71.2 ± 7.3 vs. 46.3 ± 2.7 kJ, respectively). Phosphorylation of AMPKThr172 increased by a similar magnitude (P = 0.347) immediately post INT and CON (1.6 ± 0.2 and 1.3 ± 0.3 fold, respectively; P = 0.011), before returning to resting values at 3 h post-exercise. mRNA expression of PGC-1α (7.1 ± 2.1 vs. 5.5 ± 1.8 fold; P = 0.007), VEGF (3.5 ± 1.2 vs. 4.3 ± 1.8 fold; P = 0.02) and HIF-1α (2.0 ± 0.5 vs. 1.5 ± 0.3 fold; P = 0.04) increased at 3 h post-exercise in response to INT and CON, respectively; the magnitude of which were not different between protocols.ConclusionsDespite differences in total work done, low-volume INT and CON ‘all-out’ cycling, matched for exercise duration, provides a similar stimulus for the induction of mitochondrial and angiogenic cell signalling pathways in trained skeletal muscle.

Skeletal muscle glucose uptake during treadmill exercise in neuronal nitric oxide synthase-μ knockout mice.

Nitric oxide influences intramuscular signaling that affects skeletal muscle glucose uptake during exercise. The role of the main NO-producing enzyme isoform activated during skeletal muscle contraction, neuronal nitric oxide synthase-μ (nNOSμ), in modulating glucose uptake has not been investigated in a physiological exercise model. In this study, conscious and unrestrained chronically catheterized nNOSμ(+/+) and nNOSμ(-/-) mice either remained at rest or ran on a treadmill at 17 m/min for 30 min. Both groups of mice demonstrated similar exercise capacity during a maximal exercise test to exhaustion (17.7 ± 0.6 vs. 15.9 ± 0.9 min for nNOSμ(+/+) and nNOSμ(-/-), respectively, P > 0.05). Resting and exercise blood glucose levels were comparable between the genotypes. Very low levels of NOS activity were detected in skeletal muscle from nNOSμ(-/-) mice, and exercise increased NOS activity only in nNOSμ(+/+) mice (4.4 ± 0.3 to 5.2 ± 0.4 pmol·mg(-1)·min(-1), P < 0.05). Exercise significantly increased glucose uptake in gastrocnemius muscle (5- to 7-fold) and, surprisingly, more so in nNOSμ(-/-) than in nNOSμ(+/+) mice (P < 0.05). This is in parallel with a greater increase in AMPK phosphorylation during exercise in nNOSμ(-/-) mice. In conclusion, nNOSμ is not essential for skeletal muscle glucose uptake during exercise, and the higher skeletal muscle glucose uptake during exercise in nNOSμ(-/-) mice may be due to compensatory increases in AMPK activation.

Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta

Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease, including hypertension. Its beneficial effect toward improved vasodilation results from its ability to activate AMPK and enhance nitric oxide formation in the endothelium. To date, metformin regulation of AMPK has not been fully studied in intact arterial smooth muscle, especially during contraction evoked by G protein-coupled receptor (GPCR) agonists. In the present study, ex vivo incubation of endothelium-denuded rat aortic rings with 3mM metformin for 2h resulted in significant accumulation of metformin (∼600pmoles/mg tissue), as revealed by LC–MS/MS MRM analysis. However, metformin did not show significant increase in AMPK phosphorylation under these conditions. Exposure of aortic rings to a GPCR agonist (e.g., phenylephrine) resulted in enhanced AMPK phosphorylation by ∼2.5-fold. Importantly, in metformin-treated aortic rings, phenylephrine challenge showed an exaggerated increase in AMPK phosphorylation by ∼9.7-fold, which was associated with an increase in AMP/ATP ratio. Pretreatment with compound C (AMPK inhibitor) prevented AMPK phosphorylation induced by phenylephrine alone and also that induced by phenylephrine after metformin treatment. However, pretreatment with STO-609 (CaMKKβ inhibitor) diminished AMPK phosphorylation induced by phenylephrine alone but not that induced by phenylephrine after metformin treatment. Furthermore, attenuation of phenylephrine-induced contraction (observed after metformin treatment) was prevented by AMPK inhibition but not by CaMKKβ inhibition. Together, these findings suggest that, upon endothelial damage in the vessel wall, metformin uptake by the underlying vascular smooth muscle would accentuate AMPK phosphorylation by GPCR agonists independent of CaMKKβ to promote vasorelaxation.

Muscle-derived follistatin-like 1 functions to reduce neointimal formation after vascular injury.

It is well-established that exercise diminishes cardiovascular risk, but whether humoral factors secreted by muscle confer these benefits has not been conclusively shown. We have shown that the secreted protein follistatin-like 1 (Fstl1) has beneficial actions on cardiac and endothelial function. However, the role of muscle-derived Fstl1 in proliferative vascular disease remains largely unknown. Here, we investigated whether muscle-derived Fstl1 modulates vascular remodelling in response to injury. The targeted ablation of Fstl1 in muscle led to an increase in neointimal formation following wire-induced arterial injury compared with control mice. Conversely, muscle-specific Fstl1 transgenic (TG) mice displayed a decrease in the neointimal thickening following arterial injury. Muscle-specific Fstl1 ablation and overexpression increased and decreased, respectively, the frequency of BrdU-positive proliferating cells in injured vessels. In cultured human aortic smooth muscle cells (HASMCs), treatment with human FSTL1 protein decreased proliferation and migration induced by stimulation with PDGF-BB. Treatment with FSTL1 enhanced AMPK phosphorylation, and inhibition of AMPK abrogated the inhibitory actions of FSTL1 on HASMC responses to PDGF-BB. The injured arteries of Fstl1-TG mice exhibited an increase in AMPK phosphorylation, and administration of AMPK inhibitor reversed the anti-proliferative actions of Fstl1 on the vessel wall. Our findings indicate that muscle-derived Fstl1 attenuates neointimal formation in response to arterial injury by suppressing SMC proliferation through an AMPK-dependent mechanism. Thus, the release of protein factors from muscle, such as Fstl1, may partly explain why the maintenance of muscle function can have a therapeutic effect on the cardiovascular system.

A1.35 Adiponectin isoform-mediated effects on P38 MAPK and AMPK pathways in rheumatoid arthritis synovial fibroblasts

BackgroundAdiponectin, a C1q/tumour necrosis factor homologue, was previously described to be secreted by adipocytes. Adiponectin levels were found to be increased in the synovial fluid of rheumatoid arthritis (RA) patients...

Telmisartan ameliorates insulin sensitivity by activating the AMPK/SIRT1 pathway in skeletal muscle of obese db/db mice

BackgroundTelmisartan is a well-established angiotensin II type 1 receptor blocker that improves insulin sensitivity in animal models of obesity and insulin resistance, as well as in humans. Telmisartan has been reported to function as a partial agonist of the peroxisome proliferator-activated receptor (PPAR) γ, which is also targeted by the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase (SIRT1). Here, we investigated the pathways through which telmisartan acts on skeletal muscle, in vitro as well as in vivo.MethodsNine-week-old male db/db mice were fed a 60% high-fat diet, with orally administrated either vehicle (carboxymethyl-cellulose, CMC), 5 mg/kg telmisartan, or 5 mg/kg telmisartan and 1 mg/kg GW9662, a selective irreversible antagonist of PPARγ, for 5 weeks. Effects of telmisartan on Sirt1 mRNA, AMPK phosphorylation, and NAD+/NADH ratio were determined in C2C12 cultured myocytes.Results and discussionTelmisartan treatment improved insulin sensitivity in obese db/db mice fed a high-fat diet and led to reduction in the size of hypertrophic pancreatic islets in these mice. Moreover, in vitro treatment with telmisartan led to increased expression of Sirt1 mRNA in C2C12 skeletal muscle cells; the increase in Sirt1 mRNA in telmisartan-treated C2C12 myoblasts occurred concomitantly with an increase in AMPK phosphorylation, an increase in NAD+/NADH ratio, and increases in the mRNA levels of PGC1α, FATP1, ACO, and GLUT4.ConclusionsOur results indicate that telmisartan acts through a PPARγ-independent pathway, but at least partially exerts its effects by acting directly on skeletal muscle AMPK/SIRT1 pathways.

IL-6 Indirectly Modulates the Induction of Glyceroneogenic Enzymes in Adipose Tissue during Exercise

BackgroundGlyceroneogenesis is an important step in the control of fatty acid re-esterification with PEPCK and PDK4 being identified as key enzymes in this process. We have previously shown that glyceroneogenic enzymes such as PDK4 are rapidly induced in white adipose tissue during exercise. Recent studies have suggested that IL-6 regulates adipose tissue metabolism and gene expression during exercise. Interestingly, IL-6 has been reported to directly decrease PEPCK expression. The purpose of this investigation was to determine the role of IL-6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. We hypothesized that the exercise-mediated induction of PDK4 and PEPCK would be greater in adipose tissue from IL-6 deficient mice compared to wild type controls.Methodology and Principle FindingsTreatment of cultured epididymal adipose tissue (eWAT) with IL-6 (150 ng/ml) increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK and PDK4 mRNA. AICAR decreased the expression of PDK4 and PEPCK. The activation of AMPK by IL-6 was independent of increases in lipolysis. An acute bout of treadmill running (15 meters/minute, 5% incline, 90 minutes) did not induce SOCS3 or increase phosphorylation of STAT3 in eWAT, indicating that IL-6 signalling was not activated. Exercise-induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL-6−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta-adrenergic signalling in adipose tissue from IL-6−/− mice.Conclusions and SignificanceOur findings question the role of IL-6 signalling in adipose tissue during exercise and suggest an indirect effect of this cytokine in the regulation of adipose tissue gene expression during exercise.

IL‐6 Indirectly Modulates The Induction Of Glyceroneogenic Enzymes In Adipose Tissue During Exercise

Glyceroneogenesis is an important step in the control of fatty acid re‐esterification with PEPCK and PDK4 being key enzymes in this process. It has been suggested that IL‐6 regulates adipose tissue metabolism and gene expression during exercise. The purpose of this investigation was to determine the role of IL‐6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. To achieve these goals, we utilized IL‐6 ex vivo treatment in cultured adipose tissue in combination with IL‐6−/−mice model. Treatment of cultured epididymal adipose tissue (eWAT), increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK mRNA. The activation of AMPK was independent of increases in lipolysis. An acute bout of treadmill running did not result in the induction of SOCS3 or increases in the phosphorylation of STAT3 in eWAT from WT mice. Exercise‐induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL‐6−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta‐adrenergic signalling in adipose tissue from IL‐6−/− mice. Our findings question the role of IL‐6 signalling in adipose tissue during exercise and demonstrate an indirect effect of this cytokine in the regulation of adipose tissue gene expression.This work was supported by a Bridge Funding Operating Grant from the Canadian Institute of Health Research, Institute of Nutrition, Metabolism and Diabetes to David C. Wright.

Brca1 Is Expressed And Binds To Phosphorylated Acetyl Coa Carboxylase In Skeletal Muscle

Breast cancer early onset type 1 (BRCA1) is an estrogen sensitive gene known to play a critical role in the prevention breast tumorigenesis and is also thought to contribute to DNA damage repair. Recently in breast tissue, BRCA1 was identified as a binding partner and regulator of acetyl CoA carboxylase (ACC) activity suggesting BRCA1 may act as a regulator of lipid metabolism. Currently, the role of BRCA1 has yet to be characterized in skeletal muscle. The purpose of this study was to determine if BRCA1 was expressed in skeletal muscle and if it could be immunoprecipated with the phosphorylated form of ACC (ACC-p). BRCA1 mRNA and protein was identified in skeletal muscle with the highest levels being detected in more oxidative muscles (i.e. soleus and gastrocnemius) and lower levels detected in glycolytic muscles (i.e. tibialis anterior). Further, our data suggest that BRCA1 expression in skeletal muscle is estrogen sensitive, in that BRCA1 mRNA expression was higher in female mice compared to both male and surgically ovariectomized female mice. To determine if BRCA1 could be immunoprecipated with ACC-p, acute treadmill running was utilized to induce increases in ACC-p content through exercise-induced activation of AMP-kinase. In preliminary experiments we found that female mice running at treadmill speeds of ∼27m/min for 25-30 min elicited significant increases in AMPK phosphorylation (Thr-172) and ACC-p (Ser79) content. Female C57/BL6 mice were acclimated to treadmill exercise and were then subjected to an acute bout of exercise (26.5±0.3 m/min for 28.3±2.2min). Immediately after the exercise bout, the gastrocnemius muscle was removed and snap frozen in liquid nitrogen. In response to an acute bout of treadmill exercise ACC phosphorylation (Ser 79) was increased significantly by 43% compared to the sedentary mice. No changes in total ACC were detected between groups. After samples underwent immunoprecipation with an antibody specific to BRCA1, we detected significantly more ACC-p bound to BRCA1 (176%) in the exercised group compared to the sendentary group. There were no differences detected in the total amount of BRCA1 immunoprecipitated between groups. These data indicate that in similar fashion to breast tissue, BRCA1 can bind to the phosphorylated form of ACC in skeletal muscle. These results suggest that BRCA1 is expressed in skeletal muscle and is potentially acting as a novel regulator of ACC in skeletal muscle in response to an acute bout of exercise.

Unmetabolized fenofibrate, but not fenofibric acid, activates AMPK and inhibits the expression of phosphoenolpyruvate carboxykinase in hepatocytes

Aims A lipid-lowering agent, fenofibrate, has been reported to reduce hepatic glucose production and the expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme for gluconeogenesis in the liver. However, the precise mechanisms of these effects have remained unclear. Main methods Rat hepatoma-derived H4IIE cells and murine myoblast-derived C2C12 cells were incubated with the ester form of fenofibrate and fenofibric acid, a metabolite of fenofibrate ester, and the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC), PEPCK mRNA expression and glucose production were assessed. Key findings Incubation of H4IIE hepatoma cells with the ester form of fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) and decreased PEPCK mRNA expression and glucose production. Fenofibrate-induced reductions in PEPCK expression and glucose production were abrogated by compound C, a specific AMPK inhibitor. Fenofibric acid, a metabolite of fenofibrate ester, had no effects on AMPK phosphorylation, PEPCK gene expression, or glucose production in H4IIE cells. Fenofibrate-treated mice exhibited increases in AMPK phosphorylation and a decrease in PEPCK expression in the liver but not in skeletal muscles, suggesting that unmetabolized fenofibrate accumulated and affected AMPK only in the liver. Significance These results demonstrate that fenofibrate inhibits PEPCK gene expression and hepatic glucose production in the liver via AMPK activation, even though the metabolite loses its effects on AMPK and does not work in vivo in myocytes. This novel feature of fenofibrate may provide additional benefit for the treatment of patients with disorders of both lipid and glucose metabolism.