text:
Dedicated Team
The main engagement model we use in cooperation with our clients is Dedicated Team. This is one of the more widely spread engagement models used by providers of custom software.
Envion's Dedicated Teams
Usually, a Dedicated Team is created by us after we become acquainted with the project at hand, determine the required number of team members, their roles on the team, skills sets, and level of qualifications.
Regardless of the nature of the project, we try to make sure that at least one of the team members has a good knowledge of the corresponding business domain. The team is always lead by a seasoned PM, preferably experienced in implementing similar projects.
Once the team is created and the project is underway, the client can monitor the progress whenever they like using an instant messenger, phone, email, or a project management system.
Advantages of the Dedicated Team Engagement Model
We favor the Dedicated Team engagement model over several other popular engagement models due to a number of distinct advantages, afforded by it both to our clients, and to us as the vendor implementing a project:
• The ability for the client to directly take part in selecting the members of the team
• Flexibility and the ability to quickly adjust the outsourcing arrangement in accordance with the needs of the project, including the related contract
• The ability to quickly and flexibly react to frequently changing project requirements
• All-round monitoring transparency for the client, with maximum control over the development process
• The ability to quickly hire more team members
• Full involvement of the team members in the project, and their total commitment to the client's business objectives
• A seasoned PM with relevant project delivery experience
• The ability to seamlessly integrate the project team with the client's in-house development team
• Envion provides all required project infrastructure
• Direct communication between the two project teams, or between the Envion project team, and the client's business stakeholders or technical experts
Envion-Provided Project Infrastructure
Along with our Dedicated Team, we put at our client's disposal all the required project infrastructure, including office space, workstations, networking, training, talent acquisition, VoIP, and more, thus eliminating the need for them to bear additional expenses, and then to manage several separate teams.


text:
Temperature Sensor Color Changing 15-LED Shower (Silver)
This LED shower head will change color with the temperature of the water and ensure you know by sight exactly.
For more info and to Buy, visit - https://goo.gl/UXS5Uo
Best electronic products at our electronics online shopping store at the best price with free shipping.
For more electronic products and accessories, visit - http://shrsl.com/?e4xf
Ad ID: 290246

subset: commoncrawl


text:
Ratings for the MTV Video Music Awards bounced back considerably after last year's dismal performance, giving MTV the most-watched show on TV Sunday night (Aug. 25).
The VMAs brought in 10.1 million viewers Sunday, improving by 4 million viewers over last year's show, which was the least-watched VMA telecast since 2006. It also scored the biggest audience for any show, broadcast or cable, for the night.
The telecast, driven by heavily talked-about performances from Miley Cyrus, Lady Gaga, Justin Timberlake and *NSYNC — who briefly reunited during Timberlake's mini-set — also scored big among MTV's target audience of teens and young adults. It was the No. 1 entertainment program of the year among people ages 12-34.
The awards also delivered big traffic numbers for MTV's digital and mobile properties as fans watched more than 15 million video streams — five times more than on the day of the 2012 VMAs.
MTV's red carpet show also rose significantly, more than doubling last year's viewership to 4.6 million people.


text:
Smiley wallpapersFree
Every day is different and new wallpapers! add to favorites and make your life easy with wallpaper by share on social network. Features: ? High quality images ? Full HD wallpaper, ? Supports Full HD images ? share on social networks ? You can save images in the SD card ? Free images
Downloads: 1+ (for Android)Report app for spam or abuse!
Recommended apps
Beautiful Jesus Live Wallpaper HD Free
Beautiful Jesus Live Wallpaper HD is a free, simple yet beautiful live wallpaper for you!
Michael Jackson Dancing LWP Free
Michael Jackson is showing off his moves in this free LWP of the legend!


text:
God has so much in store
And will open up a door
Sometimes it might not be what we want
Or it may be hard to trust Him
But His love is always over the brim
God knows far better than we do
And dearly loves each and everyone of you
He wants good things for all of us
You might think why if you are having a hard time
But He can use to bad and make it into a pretty rhyme
If you want to know what God's will is
Look and see what Romans 12:2 says
He doesn't want us to follow the world's ways
We need to be transformed by the renewing of our mind
Or else we we won't know and will be blind
We need to be thankful in all circumstances
And whatever God says, we need to be willing to take chances
When we do, it will help in our relationship with God
It might be lots of work and feel like you have ran lots of miles
But we will learn so much from the tests and trials
I pray that God is the One you chose to look to
As you figure out God's will for you
I ask that many doors and opportunities will open up
Also see that God can fix you up and do lots of repairs
And that you will be able to see how much God truly cares
© Copyright 2017 CalsJoy. All rights reserved.
Status: Finished
Genre: Religion and Spirituality
Short Story / Religion and Spirituality
Short Story / Other
Poem / Romance
Paste the link to picture in the entry below:
Paste the link to Youtube video in the following entry:
Cannot annotate a non-flat selection. Make sure your selection starts and ends within the same node.
An annotation cannot contain another annotation.
There was an error uploading your file.


text:
Family owned and operated boarding, training, lessons, monthly 3 phase schooling shows.
I am a rider that trains my barrel horses and that is about it and I trail ride alout and ride in the local horse clubs I will help you sell your…
We specialize in breeding quality performance horses. We stand NRHA $ producer Peppy Slidewinder, 89% NFQHA certified.
At stud: Kid With Money, palomino son of Kid Tama. Saddles, Arabians, and quarter horses for sale in Alabama and Ohio!
We are a very small private facility on 5 acres. We do rescue when space allows.
horse farm
Quality APHA & AQHA Horses. Showing, Breeding, fitting babies for Halter
we help handycap kids teach people to ride have rodeo's train horses ect.....


text:
24 April 2014
26 April 2014 Last updated at 23:46 BST
A UKIP candidate has sparked fresh controversy for his party after remarks he made on social media about the comedian Lenny Henry.
William Henwood, who is standing in local elections in north London, also made anti-Islamic comments but told the BBC he did not think his messages on social media were offensive.
The party said any breach of its rules would be investigated.
Ross Hawkins reports.


text:
On Jan. 23, The Nature Conservancy will sponsor Conservation Day at the Statehouse. The event focuses on giving community members a chance to interact with their legislators and voice their opinions and concerns on environmental issues. Dress code is business casual.
A presentation on the Indiana Conversation Alliance's 2013 legislative priorities and salient talking points is scheduled to begin at 1 p.m. in the Indiana Government Center Auditorium. (Registration begins at 12:30 p.m. in the Indiana Government Center South Atrium.)
From 2:30 p.m. – 5 p.m., advocates and lawmakers will gather in the Statehouse North Atrium for refreshments and discussion on environmental issues.
"This year is very important, because Indiana has a new governor and many new members of the legislature who need to hear about the importance of conservation of our natural environment," said organizer Angela Hughes in a news release. "Additionally, this is a budget-making session of the General Assembly, and of course we want to make sure that conservation programs are funded appropriately."
In addition to advocating for funding of the Indiana Heritage Trust, Bicentennial Nature Trust, and Clean Water Indiana programs, Hughes identified legislation to improve Central Indiana's public transit options and work toward water quality improvements through greater consumer education about issues with phosphorous in lawn fertilizers.
Lawmakers and advocates alike encourage citizens to introduce themselves to their elected representatives and communicate about the issues that they believe are important for the legislature to address and why.
At a 2011 Greening the Statehouse Forum, Ron Gifford, Central Indiana Corporate Partnership's executive vice president of public policy and a member of the Central Indiana Transit Task Force, reminded attendees that Hoosiers tend to embrace "radical incrementalism."
Before resorting to terms such as "corporate tool" or even "rat's ass Republicans" (as beloved NUVO Editor Emeritus Harrison J. Ullmann was wont to do), long-time lobbyists and Statehouse watchers suggest that when progressive agendas meet conservative ones, finding areas of common ground, such as the importance of conservation, may help identify the channels through which environmental progress can develop.
Climate, coal ash and clean energy, or "the three Cs," as the Hoosier Environmental Council's Jesse Kharbanda calls them, will be at the heart of his environmental advocacy group's air and energy priorities in 2013.
At a December meeting outlining current environmental challenges, Kharbanda encouraged his fellow advocates to acknowledge "great improvements in (Indiana's) air quality," as they pertain to EPA attainment standards, but also noted "a troubling lack of moral urgency at the state level; Indiana does not have a climate action plan."
area of concern: Indiana may be the 15th most-populated state and 38th
in terms of geographical area, but it is No. 1 in the nation for the number of
coal ash sludge lagoons.
While the state "has a great deal of self regulation by utilities, virtually every Indiana-regulated utility has had some kind of red flag raised by the EPA related to coal ash water pollution," Kharbanda said.
He advised the audience to keep an eye on Congress, where movements "to derail future federal safeguards to protect drinking water from coal ash" are underway. Finally, state lawmakers can expect to hear from HEC ideas on how the state can do more to foster clean energy, given the enormous untapped economic potential of wind, solar, and geothermal in Indiana.
Ginnye Cubel contributed to this report.


text:
A summit meeting is never just a meeting, it is also a theatrical production. As Nato leaders prepared for their summit, they envisaged at least two audiences: their own publics at home, and Russia.
Nato, it turns out, is not alone in its sense of theatre. Whether by chance or by design, Russia stole some of the limelight yesterday, when President Putin, during a trip to Mongolia, announced a ceasefire and a seven-point agreement to end the war in eastern Ukraine. The President of Ukraine, Petro Poroshenko, sent out the same news. They said there was a prospect of a peace agreement just in time to share the headlines with the fire and brimstone statements about Russian aggression likely to come out of Wales.
The question is whether the ceasefire in Ukraine is anything more than a publicity stunt; and even if it is, whether it has any chance of bringing the conflict to an end. Recent experience is not encouraging. There have been several attempts to halt the fighting, none of which have come to anything. One, negotiated by the Russia's Foreign Minister, Sergei Lavrov, and the US Secretary of State, John Kerry, was ignored by those fighting on the ground. Two months ago, the pro-Russian separatists offered a ceasefire, only to have it rejected by the government in Kiev, whose forces were then advancing. Poroshenko's hope appeared to be for a military victory that would reunite the country.
That calculation failed, partly because of the downing of the Malaysian plane in a rebel-held area, and partly because the rebels – possibly receiving more help from Russia – took back some territory and seemed ready to fight, street by street if necessary, for their remaining strongholds of Lugansk and Donetsk.
Over the past month, casualties among fighters and civilians have passed 2,500; there are now estimated to be a million refugees. The Kiev government is also receiving a more critical foreign press, with TV reports from inside the besieged cities and Human Rights Watch cataloguing atrocities on both sides.
For all these reasons – plus, perhaps pressure from the EU concerned about the economic cost of underwriting an increasingly bankrupt Ukraine – it appears that Poroshenko has plumped for trying to end the conflict. A ceasefire may suit the rebels, too, because for all the Western talk of a Russian "invasion", Russia has actually done the barest minimum to keep the rebel cause alive. If it had wanted to incorporate more territory into Russia by force, it could have done so long ago.
The Russian President is also starting to face domestic pressure from two sides: from those concerned about Russian casualties on the one hand and from those encouraged by the annexation of Ukraine to believe that a newly confident Russia could take the whole country.
So if Poroshenko wants a ceasefire, Putin wants a ceasefire, and the rebels have understood there are limits to Russian help, what could prevent the ceasefire holding? One answer concerns the divisions that have emerged on both sides of the conflict. The rebels in the east have always resembled a collection of militias, rather than a fully disciplined fighting force. There was scant welcome for the ceasefire announcement yesterday and there may well be dissidents who want to fight on.
Poroshenko, despite a strong electoral mandate, has failed to unite all political forces in a common purpose even outside the rebel east, and recently called parliamentary elections in the hope a new parliament could generate greater stability. Those of a less acceptable nationalist tendency could well oppose a ceasefire, too.
All that said, the chief obstacle to talks, let alone to an agreement, may have been overcome. Until yesterday, Poroshenko was insisting on negotiating with Russia; Putin was insisting that this was an internal Ukrainian conflict and Poroshenko should deal directly with the rebels. It remains unclear how much, if any, control Russia has ever had over the rebels. But if, as it appears, the rebels are more dependent now than they were on Russian military help, Moscow may be able to exert more influence than it could before. The next few days will show whether it can.Reuse content


text:
The Metrical Grammar of Beowulf
By (author) Calvin B. Kendall
Series edited by Simon Keynes Andy Orchard
Format: Paperback
Normal Price: $72.95
Your Price: $65.66 AUD, inc. GST
Shipping: $7.95 per order
You Save: $7.30! (10% off normal price)
Plus...earn $3.28 in Boomerang Bucks
Availability: Available to Backorder, No Due Date for Supply
Metrical Grammar of Beowulf by Calvin B. Kendall
Book DescriptionThis book argues that the formal art of the Old English epic Beowulf is shaped and determined by the poetic language which the poet inherited from the traditional, oral culture of Anglo-Saxon England. The patterns of metre and alliteration exhibited in the poem were not imposed by the poet on his language, but were part of the language which he spoke, the rules of which constituted his metrical grammar. Professor Kendall investigates the constraints of syntax, metre and alliteration which govern the formal art of Beowulf. He shows how the half-lines of the poem, which are the basic units of composition, are marked by the metrical grammar for placement in the verse clause; he also establishes conditions for the presence or absence of alliteration, which enable him to say whether in any given instance the alliterative device is a mandatory function of the rules of the metrical grammar or an option exercised by the poet. Professor Kendall alters traditional views of metre; he concludes the book with a complete index of scansion according to the rules he has established.
Buy Metrical Grammar of Beowulf book by Calvin B. Kendall from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9780521031219
(228mm x 152mm x 19mm)
Pages: 336
Imprint: Cambridge University Press
Publisher: Cambridge University Press
Publish Date: 23-Nov-2006
Country of Publication: United Kingdom
Other Editions...
Books By Author Calvin B. Kendall
Metrical Grammar of Beowulf, Hardback (June 1991)» View all books by Calvin B. Kendall
This book argues that the Old English epic Beowulf is shaped by the poetic language which the poet inherited.
» Have you read this book? We'd like to know what you think about it - write a review about Metrical Grammar of Beowulf book by Calvin B. Kendall and you'll earn 50c in Boomerang Bucks loyalty dollars (you must be a member - it's free to sign up!)
Facebook Page | Twitter | Google+ | Bulletin e-Newsletter | Blog | RSS
Become a Member | Book Lists | Recently Released | Coming Soon | Fast Delivery Books
Bestselling Books: Our Current Bestsellers | Australia's Hottest 1000 Books | Bestselling Fiction | Bestselling Crime Mysteries and Thrillers | Bestselling Non Fiction Books | Bestselling Sport Books | Bestselling Gardening and Handicrafts Books | Bestselling Biographies | Bestselling Food and Drink | Bestselling History | Bestselling Travel Books | Bestselling School Textbooks & Study Guides | Bestselling Children's General Non-Fiction | Bestselling Young Adult Fiction | Bestselling Children's Fiction | Bestselling Picture Books | Top 100 US Bestsellers
For: Schools | Organisations | Libraries | Publishers | Authors | Book Clubs | Bloggers | Affiliates
About Us | Contact Us | Help Desk | Security | Privacy | Terms of Use | Copyright
Phone: 1300 36 33 32 (9am-5pm Mon-Fri AEST) - International: +61 2 9960 7998 - Online Form
Address: Boomerang Books, 878 Military Road, Mosman Junction, NSW, 2088
© 2003-2017. All Rights Reserved. Eclipse Commerce Pty Ltd - ACN: 122 110 687 - ABN: 49 122 110 687
For every $20 you spend on books, you will receive $1 in Boomerang Bucks loyalty dollars. You can use your Boomerang Bucks as a credit towards a future purchase from Boomerang Books. Note that you must be a Member (free to sign up) and that conditions do apply.


text:
Follow on TwitterTweets by @FlowerFactor
Like Us on Facebook
Watch on YouTube
Visit the Aboutflowers.com
Channel on YouTube!
Top Flower Articles
Send Flowers!
If a friend is having a party, send a floral arrangement to her home before the party starts. She'll appreciate your personal touch.
Flowers on Flickr
Beautiful Flowers!
Fun-loving, free-spirited, playful feelings are conveyed by the whimsical floral palette, which is characterized by flowers in bold, contrasting colors. Floral arrangements featuring these hues set an upbeat tone and are ideal for birthdays, graduations, promotions and other celebrations.
© Society of American Florists
|Rose Flowers
|Flower & Plant Information and Photos - Flowers
Common names: tea rose, sweetheart rose, spray rose
Tea roses open 3 to 4 inches. Stems can be 12 — 30 inches. Spray roses can have several flowers on one stem.
Depending on the variety, roses typically last 4 — 7 days.
See also:


text:
Character overview: Simeon is an elderly gentleman, who dresses in his Sunday best. He talks slowly but vibrantly and with joy.
(Simeon hobbles onstage, carrying a thick Bible and eases himself down into a chair.)
Boy, my joints ain't what they used to be a hundred years ago, but then, not much of me's like it used to be. Except my hard head, that is. It's what's kept me pushing on all this time. And that, brothers and sisters, is a story you should hear.
Well, my name is... (ponders, then finally flips open the cover of his Bible) Simeon the Righteous. (chuckles) Amazing what you can find in this book here. Amen?
One day, I'm studying the Word and come across something entirely amazing. From... (licks his finger slowly and begins turning pages) the book of Isaiah, chapter 7, verse 14.
(clears throat) "Therefore the Lord himself will give you a sign: The virgin will be with child and will give birth to a son, and will call him Immanuel" God is with us.
"A virgin?" I says. "With child?" I says. "O Lord Almighty, what the dickens does this passage mean?"
Then by golly, the Lord answered. He said, "Well, you know what, Simeon? I'm gonna show you."
I head out that instant and tell all my friends the Lord was gonna show me a baby born from a virgin. I's excited and they's excited. So while I wait, I can't help but cry out.
(standing up weakly) "O, thank you, Lord! Ready to see your Child of Promise. 'God with us.' Yes, Lord, I'm ready. Ready and waiting. Still waiting. I'm...hey Lord, I said, I'm ready."
And I thought I had bad hearing.
Days go by, (retakes seat with a groan) then months, and years. Still no pregnant virgins. Everybody starts laughing at me, but I don't care, 'cause if in all my scripture reading I learnt one thing, it's God keeps his word, even if he don't keep our schedule. I once heard a story that God promised these people wandering the desert that they'd get a land of their own. Took forty years and that whole first generation died, but they got their Promised Land. Ha! I wonder where I heard that story.
Anyway, everybody starts laughing at me, and I just kept on trusting God. All those "friends" have passed on now, but I'm still here, by the grace of God. Thank the Lord he gave me such a hard head, right?
God'll keep his promise to me too, cuz just today, I saw her. The sweetest girl you'll ever meet. She's pregnant, and yet still a virgin too. Oh, just you wait and see. God'll pull through. And that child, whatever she's naming it, will be a reminder to everyone, God is with us.
© Copyright 2017 K D Walker. All rights reserved.
Status: Finished
Genre: Religion and Spirituality
Script / Religion and Spirituality
Script / Religion and Spirituality
Script / Religion and Spirituality
Paste the link to picture in the entry below:
Paste the link to Youtube video in the following entry:
Cannot annotate a non-flat selection. Make sure your selection starts and ends within the same node.
An annotation cannot contain another annotation.
There was an error uploading your file.


text:
World Diabetes Day
World Diabetes Day is the primary global awareness campaign focusing on diabetes mellitus and is held on November 14 each year.
Led by the International Diabetes Federation (IDF), each World Diabetes Day focuses on a theme related to diabetes, a largely preventable and treatable non-communicable disease that is rapidly increasing in numbers worldwide.[1] Topics covered have included diabetes and human rights, diabetes and lifestyle, diabetes and obesity, diabetes in the disadvantaged and the vulnerable, and diabetes in children and adolescents. While the campaigns last the whole year, the day itself marks the birthday of Frederick Banting who, along with Charles Best and John James Rickard Macleod, first conceived the idea which led to the discovery of insulin in 1922.
World Diabetes Day was launched in 1991 by the IDF and the World Health Organization (WHO) in response to the rapid rise of diabetes around the world.[1][2]
By 2016, World Diabetes Day was being celebrated by over 230 IDF member associations in more than 160 countries and territories, as well as by other organizations, companies, healthcare professionals, politicians, celebrities, and people living with diabetes and their families.[3] Activities include diabetes screening programmes, radio and television campaigns, sports events and others.
Themes of previous World Diabetes Day campaigns have focused on different factors that influence the risk of diabetes and its complications:
- 2016: Eyes on Diabetes.
- 2015: Healthy Eating.
- 2014: Go Blue for Breakfast.
- 2013: Protect our Future: Diabetes Education and Prevention.
See also[edit]
- ^ a b "Previous campaigns". World Diabetes Day. International Diabetes Federation. Retrieved 4 November 2010.
- ^ World Health Organization. Promoting health through the life-course: World Diabetes Day 2016. Geneva, accessed 7 November 2016.
- ^ Diabetes.co.uk.Diabetes Week.


text:
Also in this issue...
01 |Digital Edition02 |Front Lines03 |The Goods04 |The Goods Submissions
February 2017 Issue
Combinations: 15 Hot Picks For Spring
|Jennifer Polanz
In an issue full of containers, we had to give you something to put in all those pots. Here are some great choices for wowing customers this spring.
Nominations are open for the 2017 Green Profit/Dümmen Orange Young Retailer Award!
|Ball Publishing Staff
Nominate your favorite up-and-coming retailer today!


text:
Usually Ships in 1-5 Days
Cecilia Galante, the author of The Patron Saint of Butterflies, makes her Stepping Stones debut with a sweet series about a lovable curly-haired cupid.
Willa Bean is all wiggly with excitement. Her best friend Harper is coming for a sleepover Willa Bean wants to make Snoogy Bars and play tricks on her big sister. But Harper has a different idea. She wants to go for a night flight. Willa Bean is not okay with that. Why? Willa Bean is scared of two things . . . flying up high . . . and the dark.
About the Author
CECILIA GALANTE's YA novel, The Patron Saint of Butterflies, was a Book Sense pick, the Northeast Indie Booksellers YA Book of the Year, and a Recommended Read for Teens on Oprah's Web site. She has also written The Sweetness of Salt, Wildwood, and Hershey Herself. KRISTI VALIANT is the illustrator ofPretty Minnie in Paris, Pretty Minnie in Hollywood, theLittle Wingschapter book series, andPenguin Cha-Cha, which she wrote and illustrated. She graduated magna cum laude from Columbus College of Art & Design. Kristi lives (and dances!) in Indiana with her husband and daughters. Visit Kristi onlineKristiValiant.com."


text:
STARmeterSEE RANK
Up 111,922 this week
Mike L. Mayfield
Mike L. Mayfield was born on January 10, 1980 in Cincinnati, Ohio, USA. He is known for his work on Mr. Pickles (2013), The Rooster Project (2003) and Untitled Andre Hyland Project (2010). See full bio »
Born:in Cincinnati, Ohio, USA


text:
Mathew Williamson Runway Review: Fall 2010 was a runway showstopper at London Fashion Week
Tag Archives | fashion shows
Holly Fulton has architectural inspiration at London Fashion Week for her fall 2010 collection
Holly Fulton has architectural inspiration at London Fashion Week for her fall 2010 collectionRunway Review: Fulton's designs referenced famous world-skyscrapers including New York's Empire State Building, which reconfirmed her fascination with art-decoIn an intimate show at London Fashion Week, Royal College of Art graduate, Holly Fulton, presented her optimistic, striking Fall 2010 collection.Inspired by depictions of telephones in iconic artworks by Frenchman Louis Barillet, this collection by the Edinburgh-born designer carried a strong geometric theme, which was demonstrated through complicated, […]
Ashish Fairytale Theme Fashion Show at London Fashion Week
Every season Ashish Gupta takes us on a journey. So it comes as no surprise that on this early morning a sleepy-eyed fashion crowd were whisked away into a land of fairytales and references
Mary Katrantzou Fall Fashion Show at London Fashion Week
Mary Katrantzou Digital prints and Mary Katrantzou go hand-in-hand, but her eye for striking graphics is not her only talent as demonstrated in her Fall 2010 show
You are invited to the Armani fashion shows by way of live stream
Armani Teatro in Milan, that's the Armani Theater in English! Following suit from from the Giorgio Armani Menswear live stream fashion shows during Milan Menswear Fashion Week, we now have a bird's eye view of the women's collections during Milan Fashion Week. Make yourself a bowel of Pasta Milano, pour a glass of Barolo, and sit back and enjoy the Emporio Armani Live stream this Friday. On Saturday, a cappucino is in order to watch the Giorgio Armani Womenswear collection. […]
Frank Tell Fashion Show Fall 2010 NY Fashion Week
Runway Review: Frank Tell Fashion Show Fall 2010 NY Fashion Week. Set amongst a Dan Flavin inspired assortment of neon lights, Frank Tell's recent presentation at Milk Studios was rife with a futuristic simplicity that was appropriately less urban warrior and definitively more wearable.
Burberry Prorsum Fashion Show Fall 2010 London Fashion Week
Runway Review: After the superb trench coats of last season, you would be forgiven for questioning how Bailey could possibly out-do the designs in Burberry Prorsum's Spring collection. But step up to the challenge, he did! As the rain came down outside for London Fashion Week and a soundtrack of stormy weather blasted inside, the mood was set for British heritage brand, Burberry Prorsum's Fall 2010 show. But, despite the weather, the message certainly not one of gloom and the […]
Costello Tagliapietra Fashion Show Fall 2010 New York Fashion Week
For Fall 2010, design duo Costello Tagliapietra– Jeffrey Costello and Robert Tagliapietra– known for their immaculate takes on classic Grecian draping cited Japan and the early work of it's most iconic designers, Yohji Yamamoto and Rei Kawakubo as influences, giving their Barney's babes an assortment of polished looks that had a somewhat off-kilter, slightly relaxed and inventive zest that made for a strong outing.
How to get runway hairstyles like the Modern Geisha Updo
We always strive to be ahead of the curve and what's a better to get next season's look now than with a few simple hair styling tips and tricks?originalSeen on the runway at the Costello Tagliapietra Fall / Winter 2010 fashion show just last week, the Modern Geisha Updo could be your super glam hair look this weekend!>> Read our Costello Tagliapietra Runway ReviewAt New York Fashion Week, the Modern Geisha Updo was in order at Costello Tagliapietra for fall […]
Mulberry bags a brilliant Fall 2010 collection at London Fashion Week
In the Fall 2010 show from the much-loved British brand, Mulberry showcased a range of dazzling leather accessories as well as an impressive collection of ready-to-wear garments. Set in the beautiful location of The Orangery in Kensington Palace, Mulberry exhibited a diverse collection of 35 looks in a palette of cornflower blue, mauve, purple, orange and beige.>> Mulberry Fashion Show Photos
Mark Fast has models of all sizes on his London Fashion Week fall 2010 runway
Moments before Mark Fast's Fall 2010 London fashion Week show commenced there was only one question on everyone's lips
Marc Jacobs Fall 2010 Fashion Show New York Fashion Week
Marc Jacobs fall 2010 Runway Review: Marc Jacobs is back in full form for Fall 2010 making his collection one of the week's top highlights, if not the best


text:
Collect Each of the following items, place them in a baggie, print out the following content explanation and add to the bag. Attach a cute topper and give as a gift.
Marbles : to replace the ones you will lose during tax season
Rope : in case you get to the end of yours
Penny: to give you extra cents (sense) to know which battles to fight, & which ones to ignore.
Magic Wand : for the magician people think you should be
Piece of string : to help you "tie-up" those loose ends.
A Lifesaver : to keep you from drowning in everyday problems.
A lemon drop : to remind you that "when life gives you lemons, you make lemonade."
A lollipop : to help you lick your problems.
A rubber band : to help you to remember to be "flexible" in all things.
A Snickers candy bar : to remind you that laughter IS the best medicine.
A paper clip : to help you "hold it all together."
A stick of gum : to give you that "stick-to-it" attitude.
A safety pin : to help you "pin-point" your problems, the better to solve them
Get out of Jail Free card : for that tax return you fudged on
A mint : so you will always have a fresh outlook .
Candle : for when you're burning the midnight oil
Battery : to help you keep going and going and going.
Candy kiss : to remind you that you are loved by me


text:
Sorry I'm late guys. Here it goes.
Sorry I'm late guys. Here it goes.
Long after research contradicts common medical practices, patients continue to demand them and physicians continue to deliver. The result is an epidemic of unnecessary and unhelpful treatments.
First, listen to the story with the happy ending: At 61, the executive was in excellent health. His blood pressure was a bit high, but everything else looked good, and he exercised regularly. Then he had a scare. He went for a brisk post-lunch walk on a cool winter day, and his chest began to hurt. Back inside his office, he sat down, and the pain disappeared as quickly as it had come.
That night, he thought more about it: middle-aged man, high blood pressure, stressful job, chest discomfort. The next day, he went to a local emergency department. Doctors determined that the man had not suffered a heart attack and that the electrical activity of his heart was completely normal. All signs suggested that the executive had stable angina—chest pain that occurs when the heart muscle is getting less blood-borne oxygen than it needs, often because an artery is partially blocked.
Tucker Carlson's latest reinvention is guided by a simple principle—a staunch aversion to whatever his right-minded neighbors believe.
Tucker Carlson is selling me hard on the swamp. It is an unseasonably warm afternoon in late January, and we are seated at a corner table in Monocle, an upscale Capitol Hill eatery frequented by the Fox News star. (Carlson, who typically skips breakfast and spends dinnertime on the air, is a fan of the long, luxurious, multi-course lunch, and when I requested an interview he proposed we do it here.) As we scan the menus, I mention that I'll be moving soon to the Washington area, and he promptly launches into an enthusiastic recitation of the district's many virtues and amenities.
"I'm so pathetically eager for people to love D.C.," he admits. "It's so sad. It's like I work for the chamber of commerce or something."
Plagues, revolutions, massive wars, collapsed states—these are what reliably reduce economic disparities.
Calls to make America great again hark back to a time when income inequality receded even as the economy boomed and the middle class expanded. Yet it is all too easy to forget just how deeply this newfound equality was rooted in the cataclysm of the world wars.
The pressures of total war became a uniquely powerful catalyst of equalizing reform, spurring unionization, extensions of voting rights, and the creation of the welfare state. During and after wartime, aggressive government intervention in the private sector and disruptions to capital holdings wiped out upper-class wealth and funneled resources to workers; even in countries that escaped physical devastation and crippling inflation, marginal tax rates surged upward. Concentrated for the most part between 1914 and 1945, this "Great Compression" (as economists call it) of inequality took several more decades to fully run its course across the developed world until the 1970s and 1980s, when it stalled and began to go into reverse.
Polymath computer scientist David Gelernter's wide-ranging ideas about American life.
Last month, David Gelernter, the pioneering Yale University computer scientist, met with Donald Trump to discuss the possibility of joining the White House staff. An article about the meeting in the Washington Post was headlined, "David Gelernter, fiercely anti-intellectual computer scientist, is being eyed for Trump's science adviser."
It is hard to imagine a more misleading treatment.
By one common definition, anti-intellectualism is "hostility towards and mistrust of intellect, intellectuals, and intellectual pursuits, usually expressed as the derision of education, philosophy, literature, art, and science, as impractical and contemptible."
Here is the exchange that I had with Gelernter when I reached out to ask if he would be interested in discussing the substance of his views on science, politics and culture.
High-school textbooks too often gloss over the American government's oppression of racial minorities.
Earlier this month, McGraw Hill found itself at the center of some rather embarrassing press after a photo showing a page from one of its high-school world-geography textbooks was disseminated on social media. The page features a seemingly innocuous polychromatic map of the United States, broken up into thousands of counties, as part of a lesson on the country's immigration patterns: Different colors correspond with various ancestral groups, and the color assigned to each county indicates its largest ethnic representation. The page is scarce on words aside from an introductory summary and three text bubbles explaining specific trends—for example, that Mexico accounts for the largest share of U.S. immigrants today.
A $100 million gangster epic starring Robert De Niro, Al Pacino, and Joe Pesci has become too risky a proposition for major studios.
Martin Scorsese's next project, The Irishman, is as close as you can get to a box-office guarantee for the famed director. It's a gangster film based on a best-selling book about a mob hitman who claimed to have a part in the legendary disappearance of the union boss Jimmy Hoffa. Robert De Niro is attached to play the hitman, Al Pacino will star as Hoffa, and Scorsese favorites Joe Pesci and Harvey Keitel are also on board. After Scorsese branched into more esoteric territory this year with Silence, a meditative exploration of faith and Catholicism, The Irishman sounds like a highly bankable project—the kind studios love. And yet, the film is going to Netflix, which will bankroll its $100 million budget and distribute it around the world on the company's streaming service.
"The question confronting us as a nation is as consequential as any we have faced since the late 1940s," a group of Republican and Democratic experts write.
Ben Rhodes, one of Barack Obama's top advisers, once dismissed the American foreign-policy establishment—those ex-government officials and think-tank scholars and journalists in Washington, D.C. who advocate for a particular vision of assertive U.S. leadership in the world—as the "Blob." Donald Trump had harsher words. As a presidential candidate, he vowed never to take advice on international affairs from "those who have perfect resumes but very little to brag about except responsibility for a long history of failed policies and continued losses at war." Both men pointed to one of the Beltway establishment's more glaring errors: support for the war in Iraq.
Now the Blob is fighting back. The "establishment" has been unfairly "kicked around," said Robert Kagan, a senior fellow at the Brookings Institution and former official in the Reagan administration. As World War II gave way to the Cold War, President Harry Truman and his secretary of state, Dean Acheson, "invented a foreign policy and sold it successfully to the American people. That's what containment was and that's what the Truman Doctrine was. … That was the foreign-policy establishment." During that period, the U.S. government also helped create a system for restoring order to a world riven by war and economic crisis. That system, which evolved over the course of the Cold War and post-Cold War period, includes an open international economy; U.S. military and diplomatic alliances in Asia, Europe, and the Middle East; and liberal rules and institutions (human rights, the United Nations, and so on).
And all of them are in the temperate zone.
In late 2015, in the Chilean desert, astronomers pointed a telescope at a faint, nearby star known as a red dwarf. Amid the star's dim infrared glow, they spotted periodic dips, a telltale sign that something was passing in front of it, blocking its light every so often. Last summer, the astronomers concluded the mysterious dimming came from three Earth-sized planets—and that they were orbiting in the star's temperate zone, where temperatures are not too hot, and not too cold, but just right for liquid water, and maybe even life.
This was an important find. Scientists for years had focused on stars like our sun in their search for potentially habitable planets outside our solar system. Red dwarfs, smaller and cooler than the sun, were thought to create inhospitable conditions. They're also harder to see, detectable by infrared rather than visible light. But the astronomers aimed hundreds of hours worth of observations at this dwarf, known as TRAPPIST-1 anyway, using ground-based telescopes around the world and NASA's Spitzer Space Telescope.
Some Republicans want fewer immigrants of any stripe.
With so many other confrontations over immigration already raging, it was easy to overlook that new skirmish that Republican Senators Tom Cotton of Arkansas and David Perdue of Georgia started last week.
Just weeks into office, President Trump is embroiled in legal and political struggles over his contested travel ban on seven Muslim-majority nations and the expanded criteria for deporting undocumented migrants his administration finalized this week. Cotton and Perdue opened a new front in these escalating immigration wars by proposing legislation that would cut in half the number of legal immigrants and refugees allowed into the U.S. from today's combined level of about 1.1 million annually. Echoing Trump, Cotton insisted that high immigration levels undermined wages for working-class Americans and threatened to leave them as "a near permanent underclass."
Here's one way to confuse it.
You can tell a lot about a person from how they react to something.
That's why Facebook's various "Like" buttons are so powerful. Clicking a reaction icon isn't just a way to register an emotional response, it's also a way for Facebook to refine its sense of who you are. So when you "Love" a photo of a friend's baby, and click "Angry" on an article about the New England Patriots winning the Super Bowl, you're training Facebook to see you a certain way: You are a person who seems to love babies and hate Tom Brady.
The more you click, the more sophisticated Facebook's idea of who you are becomes. (Remember: Although the reaction choices seem limited now—Like, Love, Haha, Wow, Sad, or Angry—up until around this time last year, there was only a "Like" button.)
In a short documentary, a former KKK leader reconnects with the African American woman who helped raise him.
In a short animation, the writer describes the lifelong curiosity that led him into journalism.
In an animated interview, the author explains the problem with stereotypes.


text:
Michael Azgour | Artist
Michael Azgour is an actively exhibiting artist whose work addresses concepts related to contemporary culture. Azgour's award winning paintings have been exhibited in galleries and museums in the United States and Europe, including solo shows at Art Museum of Los Gatos and Robert Green Fine Arts in California, as well as Art Fairs such as artMRKT San Francisco, Silicon Valley Art Fair and Los Angeles Art Show. His work is part of dozens of collections, including a recent commission by Stanford's Packard Children's Hospital. Michael is a part-time faculty member at Stanford University, teaching drawing and painting courses for the Architectural Design and Continuing Studies programs. [more]


text:
customer service, compliance, Operational Risk
Dallas, TX, August 2013 – XMPro is proud to share the results achieved by Czarnikow Group Limited, after implementing XMPro iBOS (Intelligent Business Operations Suite) integrated with Microsoft CRM, Microsoft SharePoint, Infor SunSystems FMS and Allegro [Commodity Trading Platform].
Celebrating its 152nd anniversary this year, Czarnikow has grown to become a large player in the sugar and ethanol industries, supporting over 300 customers across 90 countries and dealing in roughly 10% of all sugar traded annually. With their head office in London and 11 regional offices around the world, the organization faced complex challenges with regards to managing unstructured processes, security, compliance and day-to-day operational risk.
Czarnikow has since implemented XMPro iBOS and has seen significant improvements in the following key areas:
• Managing and mitigating operational, commercial and financial risk
• Seamless links between front, middle and back office
• Strong proactive business rules and process flows to enable delegation of authority
• Underpinning the business operations to support growth and development plans
Czarnikow's ICT Manager, Stuart Durrant, says "We choose a best of breed solution, leveraging the core capabilities of the new products, but also the existing software which formed the final solution. XMPro is not only the BPM application which has allowed us to model and control our non-linear business processes, but it has also become the central point of integration between our business systems, quite simply XMPro is the glue which holds our systems together."
XMPro's Intelligent Business Operation Suite helps global companies like Czarnikow dramatically improve operational performance, compliance, customer service and harness the collective knowledge of people to work smarter, not harder.
For more information download the full case study here: http://xmpro.com/czarnikow
About XMPro
XMPro helps organizations that are dealing with unpredictable processes, which are reliant on knowledge workers, to reduce uncertainty and risk related to these processes. XMPro iBOS is a complete and integrated "intelligent business operations server" that matches business operations strategy with business process technology. Structured -, unstructured -, and hybrid processes, social, mobile and connected – all in one iBPMS solution. For more information, please visit www.xmpro.com.
About Czarnikow
Czarnikow Group Limited has been in the sugar business since 1861 and is the premier provider of world sugar market services. With a head office
in London and 11 regional offices around the world; the organization supports over 300 customers across 90 countries and deals in approximately 10% of the 50 million tons of sugar that is traded annually. Czarnikow is focused on enhancing value through the supply chain. Czarnikow offers the full range of market services and meets the differing needs of sugar producers, consumers and processors. For more information, please visit www.czarnikow.com.
Print this page
XMPro, a global provider of software and services for the Industrial Internet of Things, today announced that it has joined the GE Digital Alliance Program.
XMPro joins the list of Texas Instruments' end-to-end cloud ready partners, enabling Texas Instruments customers to easily create IoT applications using data from their TI sensors.
In the newly released white paper XMPro breaks down how the Internet of Everything is changing the way we do business.
XMPro to participate as a sponsor at the Gartner Business Process Management Summit in National Harbor, 2 – 4 April, 2013.
20 April 2011, Sydney - XMPro Pty Ltd is proud to announce their participation as a sponsor at the
XMPro congratulates Professional Advantage on their achievement of winning the XMPro Partner Awards for 2010.
Discover the best way to capitalise on your SharePoint investment with workflow or process automation.
XMPro embraces the culture of mobility.
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub


text: Hoons for Hire: The Wall of DeathDavey G. Johnson7/14/06 7:00pmFiled to: RetroHoon0EditPromoteShare to KinjaToggle Conversation toolsGo to permalink This image was lost some time after publication. Ryan over at the Jockey Journal posted this a while back, and while we thought it was cool, we hadn't clicked through until now. Shame on us. As long as there have been motor vehicles, there have been hoons. Cannon Ball Baker, Barney Oldfield, Joie Chitwood, Keiichi "The Drift King" Tsuchiya, and of course, the nameless battery of everyday hooligans who populate our "Hoon of the Day" section. Hoonage comes in myriad forms, and some of the earliest were the Wall of Death riders in England back in the '30s. Today, the tradition has been revived by some brave souls, including Ken Fox and Allan J. Ford. And while we tip our hats to these men, neither of their acts include putting a freaking lion in a sidecar and riding the wall. Tornado Smith, you may have been the bravest, looniest hoon of them all. Advertisement The Motordrome Company; Wall of Death World Tour [via The Jockey Journal]Related: More hoons [Internal]ReplyLeave a reply


text:
Saving Money on Health Care Costs
Anyone who has been to the doctor or fabricated a cruise to the emergency allowance knows that bloom affliction costs accept gotten out of control. Factor in the abandoned bulk of medication and you can see why abounding humans were determined about the allegation for bloom reform. If you wish to accumulate the bulk of your medical affliction aural a acquiescent range, again you are traveling to accept to be proactive about abbreviation the bulk you pay for medical services. Whether you accept allowance or not, actuality are a few adeptness things you can do to save money on bloom affliction costs.
Price Arcade – Bloom affliction accessories are just like any added business. Each hospital, doctor's office, and medical dispensary will accept their own agenda of fees for medical casework and the amount aberration amid them all can be significant. The hospital down the artery from you may allegation $1700 for stitches while the one that is 30 afar abroad alone accuse $1200. That is abundant of a accumulation that it would be account it to go out of your way. There are several places area you can analyze the bloom affliction costs of altered facilities. Some states accept a website you can log into. Another advantage is to acquaintance your bloom allowance provider for admonition about fee schedules.
Take Advantage of Hidden Allowances – It pays to yield a abutting attending at your bloom plan and added memberships that you may have. Sometimes you will acquisition hidden allowances that can save you hundreds of dollars on your bloom affliction costs. For example, some medical affairs accommodate admission to a assistant hotline that you can alarm any time of the day every day. With their medical experience, a assistant can be a antecedent of admonition for assorted bloom accompanying issues and can save you a cruise to the doctor. Added associates programs may accommodate abatement on medical casework such as eyes and dental care.
Change Up Your Bloom Allowance – Your bloom allowance premiums calculation appear your bloom affliction costs. You may be overpaying for your medical plan and not even apperceive it. Allowance providers are for-profit entities and amount their bloom affairs to attempt finer in the marketplace. It is a acceptable abstraction to do some amount allegory shopping. An online allowance adduce website can accomplish the action a lot easier by accouterment you with absolute time amount quotes from assorted providers. That way you can see how your plan endless and even accomplish a added frugal about-face if allegation be.
If you allegation abetment with these matters, we can help. Please appointment our website at http://www.health-insurance-buyer.com and leave your acquaintance admonition so we can acknowledge to your request.


text:
More Videos...
1970 Challenger T/A 340 Six Pack
As nice of a Challenger you will find..one owner...all original...very nice car...ck it out!!!
1962 Chevrolet Bel Air Street Rod
A 1962 Chevrolet Bel Air Street Rod I shot at the Good Guy's Nashville 2013 show...I don't have much more information than that....but it was way to cool to walk by....check it out!!...Make sure you follow me so that you don't miss any of the new videos I post daily. Apparel Provided By: http://www.etmotorgear.com Check Them Out!!
1952 International Harvester Pickup "Eight Dollar Corn" 2016 Detroit Autorama
The 1952 International Harvester L-110 Custom Pickup "8 Dollar Corn" was debuted by Korek Design at the 2016 Detroit Autorama....With Ford and Chevy pickups being Hot Rodded at a pace we have never seen before...It is cool to see someone do something different like this International Harvester L-110...It has the Ford Boss 302 under the hood....Wilwood Breaks and a 6-speed Tremec to get it all to the ground...Ryan and the boys at Korek Design hit this one out of the park...Very cool Truck...Check it out!!!...For mor coverage from Detroit Autorama please visit www.scottiedtv.com The Coolest Cars On The Web!!!
1970 Ford Torino King Cobra
1 of 3 ever made..when NASCAR got wind of this car they changed the rules..very cool..and rare..ck it out!! Thanks to Floyd Garrett's Muscle Car Museum 320 Winfield Dunn Parkway Sevierville, TN 37876-5507 http://musclecarmuseum.com/


text:
Welcome to Hotel Khusboo....
We are pleased to introduce ourselves as one of the leading Hotels of Ahmedabad-Gujarat known as walled city with its own culture, heritage, places of tourist importance and also offered us the large Industrial belt. The recently approved Mega City,
Our Hotel is situated & surrounded in Main area of Corporate Houses and the easily accessible point for all tourists and businessmen.
Distance Form
- Railway Station -04KM
- InternationalAirport-10KM
- Business District -00KM
- Gita Mandir Bus Stop -03KM
- Gandhinagar –25 kms
Our Location
"La-BellaBuglow"Shilp Cross Road,
Nr. Girish Cold Drink,
Opp. Induben Khakhrawala,
Ahmedabad-380 009


text:
Many lifestyle-related diseases are associated with low-grade inflammation and peroxisome proliferator activated receptor γ coactivator (PGC)-1α has been suggested to be protective against low-grade inflammation. However, whether these anti-inflammatory properties affect acute inflammation is not known. The aim of the present study was therefore to investigate the role of muscle PGC-1α in acute inflammation. Quadriceps muscles were removed from 10-week old whole body PGC-1α knockout (KO), muscle specific PGC-1α KO (MKO) and muscle-specific PGC-1α overexpression mice (TG), 2 hours after an intraperitoneal injection of either 0.8 µg LPS/g body weight or saline. Basal TNFα mRNA content was lower in skeletal muscle of whole body PGC-1α KO mice and in accordance TG mice showed increased TNFα mRNA and protein level relative to WT, indicating a possible PGC-1α mediated regulation of TNFα. Basal p65 phosphorylation was increased in TG mice possibly explaining the elevated TNFα expression in these mice. Systemically, TG mice had reduced basal plasma TNFα levels compared with WT suggesting a protective effect against systemic low-grade inflammation in these animals. While TG mice reached similar TNFα levels as WT and showed more marked induction in plasma TNFα than WT after LPS injection, MKO PGC-1α mice had a reduced plasma TNFα and skeletal muscle TNFα mRNA response to LPS. In conclusion, the present findings suggest that PGC-1α enhances basal TNFα expression in skeletal muscle and indicate that PGC-1α does not exert anti-inflammatory effects during acute inflammation. Lack of skeletal muscle PGC-1α seems however to impair the acute TNFα response, which may reflect a phenotype more susceptible to infections as also observed in type 2 diabetes patients.
Citation: Olesen J, Larsson S, Iversen N, Yousafzai S, Hellsten Y, Pilegaard H (2012) Skeletal Muscle PGC-1α Is Required for Maintaining an Acute LPS-Induced TNFα Response. PLoS ONE 7(2): e32222. doi:10.1371/journal.pone.0032222
Editor: Jose A. L. Calbet, University of Las Palmas de Gran Canaria, Spain
Received: October 26, 2011; Accepted: January 22, 2012; Published: February 27, 2012
Copyright: © 2012 Olesen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The present study was supported by a grant from the Augustinus Foundation, Denmark. The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National Research Foundation (# 02-512-55). CIM is part of the UNIK Project: Food, Fitness & Pharma for Health and Disease, supported by the Danish Ministry of Science, Technology, and Innovation. CIM is a member of DD2 - the Danish Center for Strategic Research in Type 2 Diabetes (the Danish Council for Strategic Research, grant no. 09-067009 and 09-075724). The Copenhagen Muscle Research Centre is supported by a grant from the Capital Region of Denmark. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The transcriptional coactivator peroxisome proliferator activated receptor γ (PGC)-1α is known to influence many aspects of metabolism and the role of PGC-1α as a master regulator of mitochondrial biogenesis and oxidative metabolism in skeletal muscle (SkM) has been confirmed repeatedly over the last decade [1]–[3]. Recently it has been suggested that PGC-1α also exerts anti-inflammatory effects. Hence overexpressing PGC-1α specifically in SkM reduces an age-associated increase in serum TNFα as well as TNFα mRNA and protein expression in SkM [4]. In accordance TNFα mRNA expression and serum TNFα increases dramatically after a single exercise bout in muscle specific PGC-1α knockout mice, but not in WT [5]. Additionally, a negative correlation has been reported between PGC-1α and TNFα mRNA levels in SkM of type 2 diabetes patients independent of BMI [6]. Taken together, these observations indicate that PGC-1α either directly or through PGC-1α mediated adaptations in SkM provides anti-inflammatory effects that potentially contribute to preventing low-grade inflammation present in many life-style related diseases [7].
Inflammatory mediators have however also been suggested to regulate PGC-1α expression [8]–[10]. Previous findings in human cardiomyocytes indicate that nuclear factor kappa light chain-enhancer of activated B cells (NFκB) binds to and inactivates PGC-1α and that TNFα enhances such NFκB mediated PGC-1α inhibition and the concomitant metabolic effects of PGC-1α [8]. In addition, a recent study shows that lipopolysaccharide (LPS) downregulates PGC-1α expression and reduces fat oxidation in mouse cardiomyocytes and mouse heart in vivo [10]. On the other hand, incubating C2C12 cells with TNFα has been reported to increase PGC-1α dependent transcriptional activity, and LPS treatment in mice has been shown to increase mitochondrial respiration when PGC-1α is elevated in SkM [9]. This suggests that PGC-1α itself is regulated during acute inflammation and these somewhat contradicting observations underline, that the role of PGC-1α in acute inflammation is not fully elucidated. Based on the findings that PGC-1α mediated adaptations in SkM protect against low-grade inflammation, it may be expected that SkM PGC-1α impairs the ability to elicit a robust acute inflammatory response.
An acute inflammatory response is a conserved mechanism highly essential for protection against invading microorganisms in all species. The response involves the induction of both pro- (TNFα, IL-6) and anti-inflammatory (IL-6 and IL-10) cytokines mediated through p38 mitogen-activated protein kinases (p38) as well as NFκB and c-Jun N-terminal kinase (JNK) signaling [11]. LPS treatment is a well-established and frequently used model to induce acute inflammation in humans and rodents [12]–[14]. Although circulating and infiltrating immune cells are seen as the traditional responders during acute inflammation, LPS stimulation has been shown to evoke p38, NFkB and JNK signaling and concomitant production of cytokines like TNFα in many tissues including SkM [9], [14], [15], which likely contributes to systemic levels during acute inflammation. This is supported by the findings that SkM functions as an endocrine organ [16] and that C2C12 cells incubated with LPS have been reported to produce and secrete IL-6 to the medium [15].
The aim of the present study was to investigate whether the level of PGC-1α expression in skeletal muscle affects an acute inflammatory response locally in skeletal muscle and systemically. This was addressed by giving LPS injections to PGC-1α whole body knockout mice (KO), muscle specific PGC-1α knockout mice (MKO) and transgenic mice overexpressing PGC-1α specifically in SkM (TG). In addition, to investigate the potential secretion of TNFα from muscle cells, primary myotubes from mouse skeletal muscle were treated with LPS.
Materials and Methods
Ethics Statement
Experiments were approved by the "Animal Experiment Inspectorate" in Denmark (permission number: 2009/561-1607) and complied with the European convention for the protection of vertebrate animals used for experiments and other scientific purposes (Council of Europe, no. 123, Strasbourg, France, 1985).
Generation and phenotypes of the whole body PGC-1α KO mice, MKO PGC-1α and TG PGC-1α mice used in the present study have been described elsewhere [1], [17], [18]. Whole body PGC-1α KO mice and littermate WT were obtained by crossbreeding of heterozygous PGC-1α KO parents. MKO and littermate WT were obtained by crossing a +/− Cre, Flox/Flox parent with a Flox/Flox parent, while TG and their littermate WT were obtained by crossbreeding of TG and WT parents. The genotypes of the mice were determined by PCR-based genotyping, as previously described [19]. Mice were kept on a 12∶12 hour light/dark cycle and had access to water and standard rodent chow ad libitum (Altromin no. 1324, Brogården, Lynge, Denmark).
Experimental protocol
Each group consisted of 10 mice with equal number of male and female mice. Ten weeks old PGC-1α KO, MKO PGC-1α, TG PGC-1α and their respective littermate WT mice were given an intraperitoneal injection of either saline as control or 0.8 µg LPS (Sigma, St Louis, MO, USA) per gram mouse, dissolved in sterile isotonic saline. Based on a preceding pilot study (data not shown) and previous studies [14], [15], [20] showing a pronounced inflammatory plasma and mRNA response 2 hours after LPS treatment, all mice in the present study were euthanized by cervical dislocation 2 hours post injection. Trunk blood was collected after decapitation and quadriceps muscles were quickly removed from all three mouse strains and frozen in liquid nitrogen. In addition visceral adipose tissue and liver were also removed from whole body PGC-1α KO mice. Samples were stored at −80°C until analyzed.
Primary cell cultures
C57BL/6 mice were euthanized by cervical dislocation and limb skeletal muscles were quickly dissected out and placed in 15 ml ice cold Dulbecco's phosphate buffered saline solution (DPBS; Invitrogen, Carlsbad, CA, USA) containing 1% glucose and 0.5% penicillin/streptomycin (Invitrogen) and placed on ice. Satellite cells were isolated and handled as previously described [21]. One day prior to the experiment, the Fusion Medium (FM) containing DMEM (Invitrogen) with 10% horse serum and 0.1% L-glutamine was exchanged with DMEM without phenol red (Invitrogen) containing 0.5% glucose, 0.05% L-glutamin and 0.1% BSA (DMEM without phenol red). Cells were used for experiments on day 8, where sufficient maturation and differentiation of the myotubes were observed. On the day of the experiment, the cells were washed in DMEM without phenol red. Half of the cell cultures (n = 6, in 3 independent experiments) were treated with 1.0 µg LPS/ml media and the other half was treated with DMEM without phenol red as control. LPS was dissolved in dimethyl sulfoxide (DMSO) and then diluted in DMEM without phenol red. After 2 hours of incubation, the medium was collected and the cells were harvested in Trizol reagent (Invitrogen). The samples were stored at −80°C until analyzed.
Cell culture media
The TNFα protein content in media from the incubated primary cell cultures was determined by Enzyme-linked immunosorbent assay (ELISA) according to the protocol of the manufacturer (eBioscience, San Diego, CA, USA). The absorption at 450 nm was determined and the TNFα protein content in the medium was calculated based on a standard curve generated from a serial diluted standard.
Plasma cytokines
Plasma was processed from the blood samples by centrifugation (2600 g for 15 min at 4°C). A MSD multiplex ELISA kit (MesoScaleDiscovery, Gaithersburg, Maryland, USA) pre-coated with antibodies against TNFα, IL-6 and IL-10 was used according to the manufacturer's protocol. MSD plates were measured on a MSD Sector Imager 2400 plate reader. Raw data were measured as electrochemiluminescence signal (light) detected by photodetectors and analyzed using the Discovery Workbench 3.0 software (MSD). A standard curve was generated for each analyte and used to determine the concentration of analytes in each sample.
RNA isolation and Reverse Transcription
Total RNA was isolated from ∼20–25 mg crushed muscle tissue, liver and adipose tissue with the guanidinium thiocyanate-phenol-chloroform method as previously described [22], [23] and in the cell culture experiment with the Trizol method following the manufacture's guidelines (Invitrogen). The final pellets were re-suspended in DEPC treated H2O containing 0.1 mM EDTA. RNA was quantified by measuring the absorbance at 260 nm.
Reverse transcription (RT) was performed using the Superscript II RNase H− system (Invitrogen) as previously described [23] and the cDNA samples were diluted in nuclease-free H2O.
Real-time PCR
Real-time PCR was performed using an ABI 7900 sequence-detection system (Applied Biosystems, Foster City, CA). Primers and TaqMan probes for amplifying gene-specific mRNA fragments were designed using the database from ensemble.org and Primer Express (Applied Biosystems). All TaqMan probes were 5′-FAM and 3′-TAMRA labeled, and primers and Taqman probes were obtained from TAG Copenhagen (Copenhagen, Denmark) except GAPDH (5′-FAM and 3′-TAMRA) and beta-actin (5′-VIC and 3′-nonflourescence), both obtained as a pre-developed assay reagent (Applied Biosystems). The sequences are given in Table 1. Real-time PCR was performed in triplicates in a total reaction volume of 10 µl using Universal Mastermix (Applied Biosystems) except TNFα converting enzyme (TACE), which was amplified using SYBGreen (Applied Biosystems). Cycle threshold (Ct) was converted to a relative amount by use of a standard curve constructed from a serial dilution of a pooled RT sample run together with the samples. For muscle samples, target gene mRNA content was for each sample normalized to single-stranded cDNA content determined by OliGreen reagent (Molecular Probes, Leiden, The Netherlands) as previously described [24]. For liver and cell culture samples, target gene mRNA content was normalized to beta-actin mRNA, which was unaffected by genotype and treatment in the respective samples and for adipose tissue samples, target gene mRNA content was normalized to GAPDH mRNA, which was unaffected by genotype and treatment in these samples.
Muscle lysate
Crushed ∼20–25 mg quadriceps muscles were homogenized in an ice-cold buffer (10% Glycerol, 20 mM Na-pyrophosphate, 150 mM NaCl, 50 mM Hepes, 1% NP-40, 20 mM β-glycerophosphate, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2 mM PMSF, 10 µg/ml Aprotinin, 10 µg/ml Leupeptin, 2 mM Na3VO4, 3 mM Benzamidine, pH 7.5) for 1 min using a tissuelyser (TissueLyser II; QIAGEN, Germany) with 30 oscillations per second. Homogenates were rotated end over end for 30 min at 4°C. The procedure with the tissuelyser and the end over end rotation was repeated. Lysates were generated by centrifugation at 16,000 g for 20 min at 4°C and collection of the protein supernatant (lysate). Protein content in lysates was measured by the bicinchoninic acid method (Thermo Scientific, Rockford, IL, USA).
SDS-PAGE and western blotting
TNFα protein as well as phosphorylation of p38Thr180, Tyr182 and p65Ser536, the active subunit of NFκB, was measured in muscle lysates by SDS-PAGE (10% or 15% Tris-HCl gel, BioRad, Denmark) and western blotting using PVDF membrane and semi-dry transfer. Twentyfive µg protein lysate was loaded for TNFα and 15 µg protein lysate for p65 and p38 phosphorylation. After the transfer, the PVDF membrane was blocked for 1 h at room temperature (TBST+5% bovine serum albumin (BSA)) and then incubated with primary antibody (1∶1000 in TBST+5% BSA) over night. Commercially available antibodies were used to detect TNFα (Cell Signaling Technology #3707), p38Thr180, Tyr182 (Cell Signaling Technology #4511) and p65Ser536 (Cell Signaling Technology #3033). The following day, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Dako, Denmark) for 1 h at room temperature (1∶5000 in TBST+5% BSA). Immobilon Western (Millipore Corporation, MA) was used as detection system and bands were visualized using an Eastman Kodak Co. Image Station 2000 MM. Band intensity was quantified using Kodak Molecular Imaging Software v. 4.0.3, and protein content or phosphorylation was expressed as arbitrary units relative to control samples loaded on each site of each gel.
Two-way ANOVA was used to test the effect of LPS and genotype within each mouse strain. Student Newman Keuls post hoc test was used to locate differences. A T-test was used to test if there was any difference in the basal levels between genotypes within each mouse strain. All values are presented as means ± S.E. A P<0.05 was considered significant.
LPS-induced TNFα responses in primary myotubes
To verify that skeletal muscle produces and secretes TNFα in response to LPS, primary myotubes isolated from C57BL/6 mice were incubated with LPS. LPS treatment increased (p<0.05) TNFα mRNA ∼30 fold and TNFα protein in the media increased (p<0.05) markedly from non-detectable levels in control cells to ∼400 pg/ml (data not shown).
Plasma cytokines
Plasma TNFα.
TG mice overexpressing PGC-1α specifically in skeletal muscle had reduced basal plasma TNFα level (0.6±0.5 pg/ml) compared with WT (2.6±1.2 pg/ml), whereas there was no difference either in whole body PGC-1α KO or in MKO PGC-1α mice compared with WT (fig. 1). In all groups, LPS increased (p<0.05) the plasma TNFα level. Whereas there was no genotype difference in the LPS-induced plasma TNFα response between whole body PGC-1α KO mice and WT, MKO PGC-1α mice had a reduced (p<0.05) response to LPS compared with WT. Furthermore, as a consequence of the lowered plasma TNFα level in TG PGC-1α mice in the basal state, a statistical interaction was observed between TG PGC-1α mice and WT. Thus the LPS-induced plasma TNFα response was higher (∼2000 fold) in TG PGC-1α mice than in WT mice (∼1000 fold).
Plasma tumor necrosis factor (TNF)α from whole body PGC-1α knockout (KO) (A), muscle specific PGC-1α KO (MKO) (B) and muscle specific PGC-1α overexpression (TG) mice (C) and their respective littermate wild type (WT) mice, 2 hours after injection with either saline (Sal) as control or lipopolysaccharide (LPS). Values are presented as means ± S.E. with n = 6–10 in each group, except WT of whole body KO strain injected with saline where n = 3, due to lack of blood. *: Significantly different from Sal within given genotype, p<0.05. #: Significantly different from WT within given treatment, p<0.05. Notice the bi-sected y-axis.
Plasma IL-6.
Basal plasma IL-6 levels were unaffected by genotype. LPS induced a ∼100–400 fold increase (p<0.05) in plasma IL-6 in all groups with no difference between genotypes (table 2).
Plasma IL-10.
Basal plasma IL-10 levels were unaffected by genotype. LPS induced a ∼20–30 fold increase (p<0.05) in plasma IL-10 in all groups with no difference between genotypes (table 2).
Basal and LPS-induced mRNA levels in skeletal muscle
TNFα mRNA.
In the basal state, whole body PGC-1α KO mice had ∼60% lower (p<0.05) TNFα mRNA content in skeletal muscle than WT and in line with this, TG mice had ∼2 fold higher (p<0.05) TNFα mRNA content than WT, whereas no significant difference was detected between MKO and WT despite a visual ∼20% reduction in MKO. LPS injections increased (p<0.05) the TNFα mRNA level ∼13–60 fold in all groups. While no genotype differences were observed in the LPS response in the whole body KO and TG strains, the TNFα mRNA level after LPS treatment was in MKO mice ∼50% lower (p<0.05) than in WT (fig. 2).
Tumor necrosis factor (TNF)α mRNA and protein in quadriceps muscle from whole body PGC-1α knockout (KO) (A,D), muscle specific PGC-1α KO (MKO) (B,E) and muscle specific PGC-1α overexpression (TG) mice (C,F) and their respective littermate wild type (WT) mice, 2 hours after injection with either saline (Sal) as control or lipopolysaccharide (LPS). Values are presented as means ± S.E. with n = 10 in each group. *: Significantly different from Sal within given genotype, p<0.05. #: Significantly different from WT within given treatment, p<0.05.
TACE mRNA.
To further examine the regulation of TNFα, a metalloproteinase, TACE, which cleaves the membrane-associated precursor of TNFα to the soluble and biological active form of TNFα, was analyzed. In general, MKO mice had 30–40% lower (p<0.05) TACE mRNA levels in SkM than WT mice, both at basal conditions and after LPS injections. No difference was observed in whole body PGC-1α KO or TG mice relative to WT. LPS had no effect on TACE mRNA levels in any of the strains (table 3).
TLR4 mRNA.
There were no differences in either basal TLR4 mRNA expression or after LPS injections in any of the strains (table 3).
IL-6 mRNA.
MKO mice had ∼50% lower (p<0.05) basal SkM IL-6 mRNA level than WT, whereas no genotype differences were observed in the basal IL-6 mRNA level in the whole body KO or TG mouse strain. In all groups, LPS induced a 130–400 fold increase (p<0.05) in IL-6 mRNA with no genotype differences (table 3).
IL-10 mRNA.
In the basal state, whole body PGC-1α KO mice had ∼55% lower (p<0.05) SkM IL-10 mRNA level than WT, whereas there were no significant genotype differences in the MKO (despite a visual 57% reduction) or TG strains. LPS increased (p<0.05) the IL-10 mRNA content ∼2–6 fold in all groups with no significant difference between genotypes (table 3).
F4/80 mNRA.
There were no genotype differences in the mRNA level of the macrophage specific marker, F4/80, in SkM of any of the strains and LPS had no effect on the F4/80 mRNA level, indicating no LPS-induced recruitment of macrophages to skeletal muscle 2 hours after LPS treatment (table 3).
TNFa protein in skeletal muscle
TNFα protein content.
TG mice had ∼9 fold higher (p<0.05) basal SkM TNFα protein level relative to WT, while there were no genotype differences in whole body KO and MKO PGC-1α mouse strains at basal conditions. LPS did not alter SkM TNFα protein content in any mouse strain, but MKO mice treated with LPS had ∼30% lower (p<0.05) TNFα protein than WT treated with LPS (fig. 2).
Basal and LPS-induced intracellular signaling in skeletal muscle
Phosphorylation of p65ser536.
Basal p65 phosphorylation was not affected by genotype in the whole body PGC-1α KO and MKO strains, but notably TG mice overexpressing PGC-1α in skeletal muscle had ∼40% higher (p<0.05) basal SkM p65 phosphorylation than WT mice. This may explain the elevated resting TNFα mRNA and protein level in these mice. LPS increased (p<0.05) p65 phosphorylation ∼30–70% in all three mouse strains. The TG mice reached ∼40% higher (p<0.05) p65 phosphorylation level after LPS treatment than WT, whereas there were no differences between whole body PGC-1α KO or MKO mice and their corresponding WT (fig. 3 and 4).
Phosphorylation of p65ser536 in quadriceps muscle from whole body PGC-1α knockout (KO) (A), muscle specific PGC-1α KO (MKO) (B) and muscle specific PGC-1α overexpression (TG) mice (C) and their respective littermate wild type (WT) mice, 2 hours after injection with either saline (Sal) as control or lipopolysaccharide (LPS). Values are presented as mean s± S.E. with n = 10 within each group. *: Significantly different from Sal within given genotype, p<0.05. #: Significantly different from WT within given treatment, p<0.05.
Representative blot of tumor necrosis factor (TNF)α protein, phosphorylation of p65 protein (p65-p) and phosphorylation of p38 (p38-p) in quadriceps muscle from whole body PGC-1α knockout (KO), muscle specific PGC-1α KO (MKO) and muscle specific PGC-1α overexpression (TG) mice and their respective littermate wild type (WT) mice, 2 hours after injection with either saline (Sal) as control or lipopolysaccharide (LPS).
Phosphorylation of p38Thr180, Tyr182.
LPS induced a similar ∼2 fold increase (p<0.05) in p38 phosphorylation in TG and WT mice, whereas no significant LPS-induced p38 phosphorylation was observed in the whole body PGC-1α KO or the MKO PGC-1α strain. No genotype differences were observed in the LPS-induced p38 phosphorylation in SkM in any of the strains (table 3).
Basal and LPS-induced mRNA levels in liver and adipose tissue from whole body PGC-1α KO mice
To examine whether PGC-1α regulates the response to acute LPS-induced inflammation in other inflammatory tissues, liver and adipose tissue mRNA was analyzed from whole body PGC-1α KO mice and WT.
TNFα and IL-6 mRNA content.
No genotype differences were observed in TNFα and IL-6 mRNA in liver or adipose tissue in the basal state. LPS induced a 6–89 fold increase (p<0.05) in TNFα and IL-6 mRNA in liver and adipose tissue with no difference between WT and KO mice (table 4).
TACE mRNA content.
There was no genotype difference in TACE mRNA level in the liver or the adipose tissueat basal conditions and no changes with LPS were observed (table 4).
IL-10 mRNA content.
In the basal state, no genotype differences were observed in IL-10 mRNA in either the liver or adipose tissue. LPS increased (p<0.05) the IL-10 mRNA level in the liver similarly in KO and WT mice. In the adipose tissue, IL-10 mRNA only increased significantly (p<0.05) in WT, whereas only visual increase (p = 0.094) were observed in PGC-1α KO mice in response to LPS treatment (table 4).
F4/80 mRNA content.
No genotype differences were observed in F4/80 mRNA content in either the liver or adipose tissue in the basal state. F4/80 mRNA was unaffected by LPS treatment in both liver and adipose tissue from WT and KO mice (table 4).
The main findings of the present study are that whole body PGC-1α KO mice had a reduced basal TNFα mRNA level and that mice overexpressing PGC-1α in skeletal muscle increased the basal TNFα mRNA and protein content in skeletal muscle, suggesting a PGC-1α mediated regulation of TNFα expression in skeletal muscle. However, basal plasma TNFα was reduced in mice overexpressing PGC-1α suggesting diminished secretion of TNFα and indicating that high TNFα levels in SkM do not lead to systemic low-grade inflammation. In addition, while high PGC-1α levels in skeletal muscle elicited a more marked LPS-induced plasma TNFα response, muscle specific knockout of PGC-1α resulted in a lower LPS-induced plasma TNFα and TNFα mRNA response, potentially reflecting a phenotype more susceptible to infections.
By use of three different PGC-1α mice models, the present study provides new insight to the role of SkM PGC-1α in basal and acute inflammation. The present findings that LPS treatment resulted in similar TNFα, IL-6 and IL-10 levels in skeletal muscle and plasma in TG and WT mice provide evidence that high levels of PGC-1α in SkM do not alter the level of inflammatory markers during acute inflammation. Of notice is however, that although the TG PGC-1α mice had a lower basal plasma TNFα level, LPS elicited a more marked fold increase in these mice. In accordance, the reduced LPS-induced TNFα response in MKO PGC-1α mice both systemically and in skeletal muscle indicates that lack of PGC-1α or PGC-1α mediated metabolic adaptations impair the ability to respond to acute inflammation. Together this suggests that high SkM PGC-1α does not exert anti-inflammatory effects during acute inflammation and SkM PGC-1α is required for a normal acute inflammatory response. Interestingly the observed responses in PGC-1α MKO and to some extent whole body PGC-1α KO mice, resemble the previously reported attenuations in LPS-induced plasma TNFα response in type 2 diabetes patients [25]. This may suggest that low levels of PGC-1α in skeletal muscle, as observed in physically inactive subjects [26] and in type 2 diabetes patients [27], impair the inflammatory response to invading pathogens.
TLR4 is the main receptor mediating LPS-induced responses in SkM. The observed similar levels of TLR4 mRNA independent of genotype and treatment indicates, that the observed lower plasma TNFα and SkM TNFα mRNA in MKO PGC-1α mice than WT in the present study are not due to a difference at the receptor level. The observed LPS-induced increase in phosphorylation of the active subunit of NFκB, p65, and to some extent p38 signaling supports that both NFκB and p38 contributed to the inflammatory response after LPS treatment in the present study. However, lack of genotype differences in the LPS-induced changes in signaling indicates that neither NFκB (p65 phosphorylation) nor p38 signaling can explain the attenuated responses in plasma TNFα and TNFα mRNA in SkM observed in MKO PGC-1α mice. Differences in NFκB or p38 signaling, earlier than 2 hour after LPS injections, of course can not be ruled out. The more marked fold increase in plasma TNFα in TG mice upon LPS treatment may on the other hand be linked to the higher p65 phosphorylation level in TG than WT. However, the unaffected TNFα protein content in TG mice in response to LPS is as such in accordance with such a mechanism as more secretion could keep the increased level constant. But the similar TNFα mRNA response in TG and WT does not support that a difference in NFκB signaling alone is responsible for the more marked plasma TNFα response in TG mice. As the LPS-induced SkM IL-6 and IL-10 mRNA and plasma levels are independent of genotype, the regulatory mediators and signaling factors involved in eliciting the observed TNFα genotype difference in MKO PGC-1α mice in response to LPS injections appear to be TNFα specific. Interestingly, the finding that MKO PGC-1α mice, both in the basal state and in response to LPS, had reduced SkM mRNA expression of the TNFα regulatory enzyme TACE [28], [29] indicates that these mice may have a decreased ability to cleave TNFα into its biological active form and thereby a reduced secretion of TNFα protein from SkM relative to WT. Such a mechanism may at least to some extent contribute to the observed impaired LPS-induced plasma TNFα response when PGC-1α is lacking in SkM.
The findings that whole body PGC-1α KO mice had lowered basal TNFα mRNA level and that TG mice had elevated basal TNFα mRNA and protein level in skeletal muscle compared with WT are in contrast to previous observations in 22 months old TG PGC-1α mice [4] and young MKO PGC-1α mice [6]. The elevated TNFα levels in skeletal muscle of TG mice in the present study may be explained by the observed higher level of basal p65 phosphorylation in skeletal muscle of TG mice. NFκB is activated by several different stress associated stimuli such as LPS and TNFα [30], but is also known to be a redox-sensitive transcription factor. An increased basal p65 activity in TG mice could possibly be due to an imbalance in ROS production and antioxidant defense, which could lead to an altered redox state in the mitochondria of these mice. However, based on enhanced superoxide dismutase 2 protein content in skeletal muscle of TG PGC-1α mice in a previous [4] and the present study (data not shown) as well as reduced protein carbonylation and oxidized nucleic acids in old TG PGC-1α mice compared with age-matched controls [4], there are no indications of elevated ROS in SkM muscle of TG PGC-1α overexpression mice. Thus, increased mitochondrial ROS levels do not seem to be the explanation for the elevated basal p65 phosphorylation and the mechanism responsible for the enhanced p65 phosphorylation in SkM of TG mice remains to be determined.
The finding that TG PGC-1α mice had elevated SkM TNFα mRNA and protein and reduced plasma TNFα relative to WT in the basal state, while there were no genotype differences in SkM and plasma for IL-6 and IL-10, again indicates a TNFα specific regulation. As TACE is required for the conversion of the TNFα precursor to circulating TNFα, a specific regulation of TACE could potentially have contributed to the observed PGC-1α dependent difference. However, the observed similar TACE mRNA levels in SkM of WT and TG mice indicate that the amount of TACE is not responsible for the observed differences between TNFα expression in SkM and systemic levels in TG mice in the basal state, although the present study cannot elucidate whether the activity of TACE is altered in these mice. Another possible explanation for the discrepancy could be that the contribution from skeletal muscle plays a minor role regarding systemic plasma TNFα levels, because TNFα released from skeletal muscle is diluted in the circulation. Cytokine production is usually believed to originate from traditional immune cells like macrophages, lymphocytes and monocytes in the blood stream and infiltrated in various tissues. Adipose tissue and the liver are also both known to respond to acute inflammation, which is also supported by the present increases in TNFα, IL-6 and IL-10 mRNA in liver and adipose tissue in response to LPS. But as skeletal muscle is the largest organ of the body and previously has been shown to produce cytokines [14], [31], [32], it may be speculated that skeletal muscle plays a significant overall role during acute inflammation. The present observation that primary myotubes incubated with LPS produced and secreted TNFα supports that skeletal muscle may be an important endocrine tissue during acute inflammation and suggests that skeletal muscle could be classified as an immunological tissue. In addition, the findings that the mRNA level of the macrophage specific marker F4/80 was unchanged with LPS treatment, suggest that the inflammatory response does not originate from newly recruited macrophages but rather resident macrophages or myofibers as suggested from the cell culture results. The magnitude by which SkM contributes to the circulating cytokine level after LPS treatment is difficult to assess and additional studies are needed to elucidate this aspect.
The present finding that overexpression of PGC-1α in skeletal muscle decreased the systemic plasma TNFα level supports a previous study showing that 22 months old TG PGC-1α mice were protected against an age-associated increase in serum TNFα [4]. Because elevated systemic TNFα has been associated with obesity [33] and type 2 diabetes [34] and TNFα is known to induce insulin resistance in vitro [35] and in vivo [36], the lowered plasma TNFα in TG mice would therefore be expected to have beneficial effects. Such a potential ability of high SkM PGC-1α to protect against systemic low-grade inflammation and at the same time posesses the ability to elicit a robust acute inflammatory response underlines that different molecular mechanism mediate these processes. Still, it may be noted, that no previous studies have reported improvements in insulin sensitivity in young TG PGC-1α mice and in fact when fed a high-fat diet for 3 weeks, TG PGC-1α mice become insulin resistant compared with WT controls [37]. In addition, the basal plasma TNFα level in the present study was relatively low (<3 pg/ml) in both TG and WT mice and therefore, the observed genotype difference in plasma TNFα may not necessarily result in metabolic differences at this age.
Finally, to elucidate the potential role of PGC-1α in the acute inflammatory response in other tissues than skeletal muscle, the liver and adipose tissue of whole body PGC-1α KO mice were examined. The observed similar LPS-induced TNFα, IL-6 and IL-10 mRNA responses in liver and adipose tissue from whole body PGC-1α KO mice and WT mice does not indicate that PGC-1α is necessary for a full inflammatory response in these tissues.
In conclusion, PGC-1α seems to be important for basal TNFα expression in skeletal muscle, potentially via effects on p65 phosphorylation. While mice overexpressing PGC-1α in SkM showed more marked plasma TNFα induction and reaching similar TNFα levels after LPS injection as WT, lack of PGC-1α and/or lack of PGC-1α mediated metabolic regulation in skeletal muscle impair a LPS-induced TNFα response both systemically and in skeletal muscle. This suggests that low levels of SkM PGC-1α could lead to an inflexible phenotype with impaired ability to cope with infections. Whether such PGC-1α associated effects are related to direct effects of PGC-1α or are secondary effects of PGC-1α mediated metabolic regulation remains to be determined.
We would like to thank Professor Bruce M. Spiegelman (Harvard Medical School, Boston, Massachusetts, USA) for the kind donation of breeding pairs to the three different PGC-1α mice strains to start breeding initially. Karina Olsen is acknowledged for skillful technical assistance.
Author Contributions
Conceived and designed the experiments: JO HP YH. Performed the experiments: JO HP . Analyzed the data: JO HP SL SY NI. Contributed reagents/materials/analysis tools: HP YH. Wrote the paper: JO HP.
- 1. Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, et al. (2002) Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature 418: 797–801. 10.1038/nature00904 [doi];nature00904 [pii].
- 2. Wende AR, Schaeffer PJ, Parker GJ, Zechner C, Han DH, et al. (2007) A role for the transcriptional coactivator PGC-1alpha in muscle refueling. J Biol Chem 282: 36642–36651. M707006200 [pii];10.1074/jbc.M707006200 [doi].
- 3. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, et al. (1999) Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98: 115–124. S0092-8674(00)80611-X [pii];10.1016/S0092-8674(00)80611-X [doi].
- 4. Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, Moraes CT (2009) Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging. Proc Natl Acad Sci U S A 106: 20405–20410. 0911570106 [pii];10.1073/pnas.0911570106 [doi].
- 5. Handschin C, Chin S, Li P, Liu F, Maratos-Flier E, et al. (2007) Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1alpha muscle-specific knock-out animals. J Biol Chem 282: 30014–30021. M704817200 [pii];10.1074/jbc.M704817200 [doi].
- 6. Handschin C, Choi CS, Chin S, Kim S, Kawamori D, et al. (2007) Abnormal glucose homeostasis in skeletal muscle-specific PGC-1alpha knockout mice reveals skeletal muscle-pancreatic beta cell crosstalk. J Clin Invest 117: 3463–3474. 10.1172/JCI31785 [doi].
- 7. Handschin C, Spiegelman BM (2008) The role of exercise and PGC1alpha in inflammation and chronic disease. Nature 454: 463–469. nature07206 [pii];10.1038/nature07206 [doi].
- 8. Alvarez-Guardia D, Palomer X, Coll T, Davidson MM, Chan TO, et al. (2010) The p65 subunit of NF-kappaB binds to PGC-1alpha, linking inflammation and metabolic disturbances in cardiac cells. Cardiovasc Res 87: 449–458. cvq080 [pii];10.1093/cvr/cvq080 [doi].
- 9. Puigserver P, Rhee J, Lin J, Wu Z, Yoon JC, et al. (2001) Cytokine stimulation of energy expenditure through p38 MAP kinase activation of PPARgamma coactivator-1. Mol Cell 8: 971–982. S1097-2765(01)00390-2 [pii].
- 10. Schilling J, Lai L, Sambandam N, Dey CE, Leone TC, et al. (2011) Toll-like Receptor-mediated Inflammatory Signaling Reprograms Cardiac Energy Metabolism by Repressing Peroxisome Proliferator-activated Receptor {gamma} Coactivator-1 (PGC-1) Signaling. Circ Heart Fail. CIRCHEARTFAILURE.110.959833 [pii];10.1161/CIRCHEARTFAILURE.110.959833 [doi].
- 11. Carpenter S, O'Neill LA (2009) Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signalling proteins. Biochem J 422: 1–10. BJ20090616 [pii];10.1042/BJ20090616 [doi].
- 12. Andreasen AS, Krabbe KS, Krogh-Madsen R, Taudorf S, Pedersen BK, et al. (2008) Human endotoxemia as a model of systemic inflammation. Curr Med Chem 15: 1697–1705.
- 13. Favorite GO, Morgan HR (1942) Effects produced by the intravenous injection in man of a toxic antigenic material derived from eberthella typhosa: Clinical, hematological, chemical and serological studies. J Clin Invest 21: 589–599. 10.1172/JCI101337 [doi].
- 14. Frost RA, Nystrom GJ, Lang CH (2002) Lipopolysaccharide regulates proinflammatory cytokine expression in mouse myoblasts and skeletal muscle. Am J Physiol Regul Integr Comp Physiol 283: R698–R709. 10.1152/ajpregu.00039.2002 [doi].
- 15. Frost RA, Nystrom GJ, Lang CH (2003) Lipopolysaccharide and proinflammatory cytokines stimulate interleukin-6 expression in C2C12 myoblasts: role of the Jun NH2-terminal kinase. Am J Physiol Regul Integr Comp Physiol 285: R1153–R1164. 10.1152/ajpregu.00164.2003 [doi];00164.2003 [pii].
- 16. Brandt C, Pedersen BK (2010) The role of exercise-induced myokines in muscle homeostasis and the defense against chronic diseases. J Biomed Biotechnol 2010: 520258. 10.1155/2010/520258 [doi].
- 17. Geng T, Li P, Okutsu M, Yin X, Kwek J, et al. (2010) PGC-1alpha plays a functional role in exercise-induced mitochondrial biogenesis and angiogenesis but not fiber-type transformation in mouse skeletal muscle. Am J Physiol Cell Physiol 298: C572–C579. ajpcell.00481.2009 [pii];10.1152/ajpcell.00481.2009 [doi].
- 18. Lin J, Wu PH, Tarr PT, Lindenberg KS, St-Pierre J, et al. (2004) Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice. Cell 119: 121–135. 10.1016/j.cell.2004.09.013 [doi];S0092867404008864 [pii].
- 19. Leick L, Wojtaszewski JF, Johansen ST, Kiilerich K, Comes G, et al. (2008) PGC-1alpha is not mandatory for exercise- and training-induced adaptive gene responses in mouse skeletal muscle. Am J Physiol Endocrinol Metab 294: E463–E474. 00666.2007 [pii];10.1152/ajpendo.00666.2007 [doi].
- 20. Huey KA, Meador BM (2008) Contribution of IL-6 to the Hsp72, Hsp25, and alphaB-crystallin [corrected] responses to inflammation and exercise training in mouse skeletal and cardiac muscle. J Appl Physiol 105: 1830–1836. 90955.2008 [pii];10.1152/japplphysiol.90955.2008 [doi].
- 21. Kusuhara K, Madsen K, Jensen L, Hellsten Y, Pilegaard H (2007) Calcium signalling in the regulation of PGC-1alpha, PDK4 and HKII mRNA expression. Biol Chem 388: 481–488. 10.1515/BC.2007.052 [doi].
- 22. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159. 10.1006/abio.1987.9999 [doi];0003-2697(87)90021-2 [pii].
- 23. Pilegaard H, Ordway GA, Saltin B, Neufer PD (2000) Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279: E806–E814.
- 24. Lundby C, Nordsborg N, Kusuhara K, Kristensen KM, Neufer PD (2005) Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies. Eur J Appl Physiol 95: 351–360. 10.1007/s00421-005-0022-7 [doi].
- 25. Andreasen AS, Pedersen-Skovsgaard T, Berg RM, Svendsen KD, Feldt-Rasmussen B, et al. (2010) Type 2 diabetes mellitus is associated with impaired cytokine response and adhesion molecule expression in human endotoxemia. Intensive Care Med 36: 1548–1555. 10.1007/s00134-010-1845-1 [doi].
- 26. Alibegovic AC, Sonne MP, Hojbjerre L, Bork-Jensen J, Jacobsen S, et al. (2010) Insulin resistance induced by physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young men. Am J Physiol Endocrinol Metab 299: E752–E763. ajpendo.00590.2009 [pii];10.1152/ajpendo.00590.2009 [doi].
- 27. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, et al. (2003) PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34: 267–273. 10.1038/ng1180 [doi];ng1180 [pii].
- 28. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, et al. (1997) A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385: 729–733. 10.1038/385729a0 [doi].
- 29. Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, et al. (1997) Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385: 733–736. 10.1038/385733a0 [doi].
- 30. Barnes PJ, Karin M (1997) Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066–1071. 10.1056/NEJM199704103361506 [doi].
- 31. Borge BA, Kalland KH, Olsen S, Bletsa A, Berggreen E, et al. (2009) Cytokines are produced locally by myocytes in rat skeletal muscle during endotoxemia. Am J Physiol Heart Circ Physiol 296: H735–H744. 01309.2008 [pii];10.1152/ajpheart.01309.2008 [doi].
- 32. Pedersen BK, Febbraio MA (2008) Muscle as an endocrine organ: focus on muscle-derived interleukin-6. Physiol Rev 88: 1379–1406. 88/4/1379 [pii];10.1152/physrev.90100.2007 [doi].
- 33. Hotamisligil GS, Shargill NS, Spiegelman BM (1993) Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259: 87–91.
- 34. Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444: 860–867. nature05485 [pii];10.1038/nature05485 [doi].
- 35. Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF, et al. (1996) IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance. Science 271: 665–668.
- 36. Plomgaard P, Bouzakri K, Krogh-Madsen R, Mittendorfer B, Zierath JR, et al. (2005) Tumor necrosis factor-alpha induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes 54: 2939–2945. 54/10/2939 [pii].
- 37. Choi CS, Befroy DE, Codella R, Kim S, Reznick RM, et al. (2008) Paradoxical effects of increased expression of PGC-1alpha on muscle mitochondrial function and insulin-stimulated muscle glucose metabolism. Proc Natl Acad Sci U S A 105: 19926–19931. 0810339105 [pii];10.1073/pnas.0810339105 [doi].