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Rating: 7.5/10 1,490 votes
Runtime: 130 min
Language: French
Country: France
Color: Color
IMDb Link: http://www.imdb.com/title/tt0086655/
Director: Jean Becker
Isabelle Adjani
Alain Souchon ... Fiorimonto 'Florimond' Montecciari - dit 'Pin-Pon'
Suzanne Flon ... Nine dite 'Cognata'
Jenny Clève ... Madame Montecciari - la mère de Pin Pon
Maria Machado ... Paula Wieck Devigne dite 'Eva Braun' - la mère d'Eliane
Evelyne Didi ... Calamité
Jean Gaven ... Leballech - le patron de la scierie
François Cluzet ... Mickey
Manuel Gélin ... Boubou
Roger Carel ... Henri dit 'Henri IV'
Michel Galabru ... Gabriel Devigne - le père d'Eliane
Marie-Pierre Casey ... Mademoiselle Tussaud - la garde-malade
Cécile Vassort ... Josette
Edith Scob ... La doctoresse
Fuelled by a scorchingly erotic performance from Isabelle Adjani, the ingeniously plotted "One Deadly Summer" spirals from provincial drama into a disturbing and complex psychological thriller, proving itself among the most under-rated of modern French films. In the hottest summer since Body Heat, Eliane (Adjani) and local mechanic Pin Pon (Alain Souchon) begin an affair then marry. From this starting point (a similar plot device to Chabrol's Le Boucher, 1969), life in a beautiful small town in Southern France begins to come apart. Under Jean Becker's direction every character is fully rounded, and the naturalistic tone adds considerably to the impact of an intense drama that offers the perfect showcase for Adjani, establishing once and for all what an extraordinarily accomplished actress she can be. Where Beatrice Dalle in Betty Blue was similarly erotically charged and mentally unstable, Eliane has a dark and obsessive agenda that anticipates the sexual psychosis of Basic Instinct.
Combining an art-house love of the French countryside with more class than a dozen Hollywood erotic thrillers, "One Deadly Summer" is a striking vision of sex gone bad which builds to a shocking climax. It deserves to take its place as a modern classic.
Screenshots :
Download Links:
L été meurtrier (1983) Aka One Deadly Summer
7 comments:
- Raymond said...
Subtitles will soon be available. 80% completed. Will post here.
- June 30, 2010 at 3:31 PM
- Raymond said...
Just finished subs.
- July 5, 2010 at 11:07 AM
- galmuchet said...
- This comment has been removed by the author.
- July 5, 2010 at 11:38 AM
- galmuchet said...
Pretty nice quality version (maybe it's OK with your subs) in MKV here --> http://www.foriegnmoviesddl.com/search?q=L%27Et%C3%A9+meurtrier
- July 5, 2010 at 11:42 AM
- Raymond said...
Sorry Galmuchet, I didn't realise that subs were already included in your upload. There was no mention of them in your write-up. I could have saved a lot of wasted time. Thanks for your great uploads that keep coming and coming, and your "Duck is Dead".
- July 6, 2010 at 10:18 AM
- galmuchet said...
to Raymond ... there is no sub in MKV version I spoke about your subtitles ...
- July 6, 2010 at 10:43 AM
- Raymond said...
My download of your movie has subs???
As a point of information, my subs were written to your movie version.
- July 21, 2010 at 11:21 AM
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|Half Baths
|116 m²
|Property type
|Minimum Stay
|2 - 999 nights
This creekside chalet is located right next door to the Creekside Lodge, pool, and hot tubs. With two private bedrooms and a sofa sleeper, it can easily accommodate eight guests. Relax on the private deck and take in the mountain air and the sound of the creek below. With rustic interior decor and a view of Mt. Hood Skibowl's illuminated slopes, this chalet will enhance your lodging experience. Amenities include Hi-Speed wireless internet, cable plasma TV with HBO, DVD, VCR, board games, Xbox games, grill on the deck, and a bench in the garage for ski and snowboard gear. You'll have to leave Fido at home though because this unit is not pet-friendly.
Located in the heart of Government Camp, Collins Lake Resort® features a year-round outdoor heated swimming pool, hot tub, leisure pool, and sauna! Enjoy being walking distance to Government Camp shops and restaurants.
Only when you book directly through us at Collins Lake Resort® can you enjoy the many local discounts we offer through our Gold VIP program and our ON-SITE concierge to make sure any needs that arise during your stay are addressed promptly. Our ON-SITE concierge can assist you with pre-arrival grocery stocking, exclusive area discounts, and more to make your vacation as comfortable as possible.
CURRENT SPECIALS - Restrictions apply, not valid during holidays
• STAY LONGER & PAY LESS – Make sure you have plenty of time to enjoy all of the beauty and FUN we have to offer on the mountain, stay 5 or more nights and the nightly rate is automatically reduced.
• Visiting for a Wedding at Mt. Hood Skibowl? Call us directly to get your discount applied!
• 10% off Veterans/ Active Duty – Call directly to apply this discount.
• SLIDE FREE MIDWEEK – Summer 2016! See below for package details.
**SLIDE FREE MIDWEEK** Stay midweek and slide free at Mt. Hood Skibowl Adventure Park! Stay for a minimum of two nights between Sunday and Thursday and receive two free slides on the famous Alpine Slide at Mt. Hood Skibowl Adventure Park. Slides are valid Monday through Friday. Restrictions Apply – Ask for details!
SUMMER VIP DISCOUNTS, Collins Lake Resort® During the Summer months enjoy our exclusive discounts for Mountain Bike Rentals, Kayak, Canoe and Paddle Board rentals through Mt. Hood Adventure, Guided Hiking tours, Whitewater Rafting tours, local Spa Services, local Dining Discounts, and local Retail Discounts.
** SKI FREE MIDWEEK** Stay midweek and ski free at Mt. Hood Skibowl & Timberline! Stay for a minimum of two nights between Sunday and Thursday and receive two adult open-close lift tickets per day per ski area for Skibowl & Timberline. Redeem up to 2 tickets per ski area per day. Tickets are valid Monday through Friday. Restrictions Apply – Ask for details!
WINTER VIP DISCOUNTS, Collins Lake Resort® guests have access to DISCOUNTED LIFT TICKETS & Tubing Tickets ON-SITE to Mt. Hood Skibowl, Timberline Lodge, and Mt. Hood Meadows. You will also have access to exclusive discounts for Rental equipment, lessons, tuning services, spa services, tubing, COSMIC Tubing, retail and activities through Mt Hood Adventure!
If you'd like to BOOK NOW or view our VIP Discounts, view the links within this listing! Or call us at 800-234-6288
This Chalet is located in the heart of Government Camp. During the winter guests enjoy the short walk to Mt. Hood Skibowl, where they can ski all day and all night! There are also two other Ski Resorts just a few miles away. During the summer enjoy the hiking trails that will take you to beautiful Mirror Lake, Trillium Lake, or maybe a waterfall. If you need help planning your adventures, just ask our on-site Concierge.

subset: commoncrawl


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Eddie possessed a sexiness that is real. With a list of attributes are endless. Beautiful face, piercing eyes, mouth-watering cock, great balls and one fine lickable ass. He is a exhibitionist who loves to get naked and show off his hot toned body. Along with his big hard cut cock.
Speaking of which, that cock. Is that perfection of what? The mushroom head is just perfect! Imagine that dick pounding your ass. But for now you will all have to settle for Eddie jerking it off ever so slowly. With the pre-cum just oozing out. He eventually cums and does he ever. Eddie has a great future because he is like fine wine. He will just get better with age.


text: Maintree Create or update sensor page To add new tabs: Create tab: descendantsCreate tab: pictures → (Charles L Timberlake (?), 1) → (Randall Timberlake (?), 2)→→ (Justin Randall Timberlake (1981), 3) Charles L Timberlake (?)/infoCharles L Timberlake (?)/sensorCharles L Timberlake (?)/tree Retrieved from "http://familypedia.wikia.com/wiki/Charles_L_Timberlake_(%3F)/sensor?oldid=369116" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Category: Sensor pages


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Library to host sale
The annual Becker County Friends of the Library Crazy Days Book Sale is Tuesday from 9 a.m. to 3 p.m.
While you are out enjoying the Detroit Lakes Crazy Daze activities, stop by the library to get the great book bargains.
Becker County Friends of the Library will be selling books, audio books, CDs, and DVDs in the main meeting room of the library. Proceeds from this event help sponsor library programs.


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Basic Steak Sandwich
READY IN: 35mins
A simple but really tasty steak sandwich. Feel free to use which ever type of steak or bread you like. Also you may cook the steak any way you like. I like to grill mine for 12 minutes on low to get a nice medium rare steak.
Top Review by Palis Favorites
Yummmmy! It was simple but had enough flavor for us. Had to add a little lemon juice because my husband cant eat his steak without it, but other than that this was great. Made for Ramadan Tag.
See Discussion:2 Reviews
Ingredients Nutrition
- 1 sirloin steak (cooked medium rare, but you can cook it as you like)
- 1 tablespoon barbecue sauce
- salt
- pepper
- 1 yellow onion (small)
- 1⁄2 lb mushroom
- vegetable oil (you may use your preferred cooking oil if you don't like vegetable oil)
- bread (I like using 6-inch toasted sub)
Directions See How It's Made
- Once your stake is grilled, slice it into thin strips.
- Place bbq sauce a little salt and a little pepper in a bowl.
- Add the steak to the bowl and mix the ingredients together until the steak is coated well. You may add more sauce if you need to.
- Peel the onion; slice thinly.
- Slice mushrooms into medium sized slices.
- Turn stove to high.
- Place a large skillet on the stove.
- Add mushrooms and onions and a few table spoons of cooking oil.
- Stir while cooking; the mushrooms and onions should become soft and slightly brown.
- Add the mushrooms and onions to the open sub.
- Place the strips of steak on top.
- I like to drizzle a little ketchup on top, but thats up to you.
- Enjoy!


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Poems: Just Go & How we Renaissed
Books & Poetry
Reflections both metaphysical and related to the Renaissance inspire Span Hanna's two poems in this week's Poet's Corner.
Just Go
Some say that nothing can be known for sure,
suggesting all we think we know is wrong.
The power of the argument is strong,
but that's the measure of it – nothing more.
It sets no obligation to ignore
our skills of observation. They belong
to us intrinsically, as art and song
have shown: we humans at our very core.
So if the world appears to be mundane
and all the best is stale, or somewhat droll,
then learn as if there's everything to learn.
Whatever you discover, more remains,
as if you went out one day for a stroll
and just kept walking, never to return.
How we Renaissed
Some like to think it starts with Gutenberg,
as if the mere invention of his press
were all it took to wipe the medieval
world away – edited, you might say,
down to a footnote. Why not? Who's to claim
it wasn't in the surprise domestication
of the word, except for the simple fact
that most could still not read it, yet? But had
their eyes, quite good enough to see emerge
the paintings, sculptures… Not to mention ears
to hear the new thoughts argued, if obliging
the mind to plough with a hardier kind of ox
the fields of enquiry brought now into play,
and in its quizzical furrows sow not seed
but some fresh hybrid, sprung from ancient grain.
Span Hanna is a former South Australian who has been living in Victoria since 2001. He is the editor of an English-language fortnightly publication (Modern Asian), provided free to the Asian community in Victoria. He worked as a teacher for more than 20 years, and lived in China for four years. See two more of Hanna's poems in last week's Poet's Corner.


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After disclosing that its officers accompanied Apple on a search for a lost iPhone prototype, the San Francisco Police Department told CNET that it's launching an investigation into the matter. Officers from the SFPD went with Apple after the company tracked a lost prototype of the iPhone to a home in San Francisco's Bernal Heights neighborhood. According to a Saturday press release from the department, officers did not enter the home to search for the device, though Apple employees did.
Apple declined to file a report about the search, which is why there had not been a previous record of the event, the department said.
The search did not turn up the device. According to CNET, the man whose home was searched, Jose Calderon, said he is "talking to attorneys" but did not say what he was talking with them about.
The SFPD did not immediately return a request to confirm the CNET report.
In other iPhone 5 news, a picture posted to Flickr by an Apple software engineer and reposted by PocketNow may have been taken with an iPhone that has an 8 MP camera. The next iPhone is rumored to be packing an 8MP camera sensor, which means the photo has sent Apple fans into a tizzy.
This is my next pointed out in its report on the photo that the coordinates on the embedded data match up with Apple's Cupertino, Calif., headquarters, though it's worth noting that it's fairly easy to fake photo data. A fun, if unbelievable tidbit: One of TIMN's dedicated readers actually uncurved the reflection in the plate to reveal something that looks like — but could not be — the reflection of someone taking a picture of the plate with a phone.
Clearly, Apple just needs to release this thing already to put us all out of our speculative misery.
Related stories:
More technology coverage from The Post
Lost iPhone: SFPD escorted Apple to search man's home


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Discount Outdoor Patio Furniture
Sunday, February 26th, 2017
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Friday, February 24th, 2017
Sunday, March 5th, 2017


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The North Coast Missioners, is an undergraduate secret society at Southern Cross University , Lismore, New South Wales. Traditionally peered with other secret societies including Yale. The society's alumni organisation, is the Trust Balzer Association, named by Thomas Watts, who co-founded The North Coast Missioners. The society is formally known as 'hanging out' and members known as 'missioners'.
North Coast Missioners at Wikia


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Eloy Vargas
Eloy Vargas
Image source bigbluefans4uk.com
Eloy Antonio Camacho Vargas was born on December 30th, 1988 in the city of Moca in the Espaillat Province of Dominican Republic.
His mother Ana Luisa Vargas and his father Eloy Camacho Lantigua who passed away in 2010 had him and his three sisters Eloisa, Marleny and Hania in the Los Lopez neighborhood in the city of Moca.
Since his father's passing on November 9th, 2010 he always honors him through his social network profiles.
Through his participation in the minors category in the Don Bosco Clubs in Moca and in the junior Club Domingo Paulino in Santiago, Vargas obtained a scholarshipat American Heritage in Miami, Florida at the age of 15. He studied there until 2008 when he finished Highschool and went on to Miami Dade College 2009-2010. He then went to Kentucky University to play in the NCAA for the Wildcats where he stayed for two seasons making it to the finals in 2011 and Champion in 2012.
In 2012 he came to play for the Dominican National League (LNB), but he suffered an injury to his meniscus requiring surgery on October 2012 and after physical therapy of aproximately 8 months he went on to dispute the last two months of the season, anxious to get back in the game. He played for the New York Nicks Summer League averaging 5.6 points, 3.8 rebounds, and 15.8 minutes in five games.
He helped the Dominican National Team qualify for the FIBA Basketball World Championship 2014 in Spain, and has signed to play professional basketball for the Fuenlabrada team in Madrid, Spain.


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Monday, April 2, 2012
Patience Pays Off
Like Amazon, you can click on the book title, and an overview of the book appears.
If you don't have a Nook, Remember my books are available on Kindle here: click here
One nice thing about Amazon is that you can click on the book, and it will let you peek inside for a sample!
Don't have a Kindle? You can download an free application that allows you to buy and read Kindle books on your PC, Ipad, or Iphone. Click here to learn more.
Thanks to those who've already purchased my books....there will be more, soon.
My latest is available both on Nook and Kindle!
It includes some of my favorite (and my readers' favorite) posts, such as My Restroom Rant, Time for a Nooner, and the Pleasure Hunt.
If you like my blog and my books, tell your friends; if you've already bought one or more of my books, go back and post a review. I'd appreciate it!
Okay, that's enough shameless self-promotion for one day!
Love you all!
12 comments:
- Vodka Logic said...
Very cool. I will definitely check them out
- April 2, 2012 at 3:05 AM
- River said...
I'm still deciding what type of e-reader to get....but I do have an ever-lengthening list of e-books to buy.
- April 2, 2012 at 5:04 AM
- Brian Miller said...
that is awesome eva....excited for you...
- April 2, 2012 at 7:56 AM
- A Beer for the Shower said...
I love shameless promotion as much as the next writer. I don't have a Nook... nor do I have a Kindle... but I do have that Kindle Cloud reader program installed. I'm off to Amazon to check it out!
- April 2, 2012 at 9:17 AM
- rosaria williams said...
- April 2, 2012 at 10:45 AM
- Terri Sonoda said...
FAbulous! Congratulations Eva! I'm hoping to self-publish soon. I may have to pick your brains when the questions come up.
Have a super Monday! Huggsss
- April 2, 2012 at 10:48 AM
- Sue said...
Glad you finally prevailed!
- April 2, 2012 at 11:25 AM
- Carol Wyer said...
Downloaded and eagerly waiting to read it Eva.
I'll promote it on my facebook page for you too.
- April 2, 2012 at 12:15 PM
- Al Penwasser said...
You gave Nook a look? Congratulations! How cool is that? How'd you do it, how'd you do it? Would you be interested in helping out a fellow American who's down on his luck? You know you're my inspiration, don't you?
- April 2, 2012 at 4:05 PM
- The Frisky Virgin said...
This is really so awesome. I was just talking with my mom about Kindles. I'm more of a traditionalist, but I'm beginning to see some major benefits of the Kindle. It's a major battle between the traditionalist, bookstore-loving me and the Kindle-interested me. You can bet I'm buying these when I get one.
- April 2, 2012 at 9:10 PM
- Eva Gallant said...
I was a holdout for a long time. I finally bought the $139 one Saturday, and I think I'm going to like it.
P.S. If you're enjoying Country Connections, you may want to buy that kindle or get the free app soon! (hint, hint)
- April 2, 2012 at 11:00 PM
- Unknown Mami said...
Well done, Eva!
- April 3, 2012 at 6:05 PM


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Data are provided for 49 species of arable weeds respecting their approximate average seed production and frequencies which indicate the absence of correlation between them.
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text:
Arthritis is a very painful disease that afflicts millions of people. Just as it is with other medical conditions, you should research and learn about arthritis and the various treatments that may help relieve some of the symptoms. The below article provides lots of tips for treating this condition.
If you are diagnosed with rheumatoid arthritis, do not give up on the activities and hobbies that you enjoy. Although some activities may instigate your arthritis, there may be ways to adapt them so that you can continue to enjoy them without pain. Find ways to make your hobbies easier and less stressful.
Take control of the situation by educating yourself about your condition. You can find a lot of information about managing pain, exercise, and nutrition from a variety of sources. As research progresses, you can also find new treatments and lifestyle changes that can keep your pain under control.
Electrical stimulation can be a great treatment for osteoarthritis, but consult your doctor about the pros and cons. It has been shown that these treatments work very well at reducing both pain and swelling in the knees that is caused by arthritis.
Learn how to maintain your posture to make your joints healthier and stronger. This can minimize some of the pain caused by arthritis. Refrain from slouching in your chair and stand up straight when you are walking and exercising. Distribute your weight evenly on each leg. Proper posture will make your joints and your back feel better, and may reduce your arthritis symptoms.
Laughter is one way to aid in alleviating stress and pain, and it can start certain chemicals, like endorphins, flowing through your bloodstream that can help alleviate the inflammation. Laughter is very good medicine, and it's absolutely free!
Even a modest amount of weight loss can improve the symptoms of arthritis. Weight can influence the effects that arthritis has on you, and losing weight could provide a huge reduction of arthritis pain. A few pounds could make a huge difference in the pressure applied to your joints.
After you have exhausted all methods of arthritis treatment without success, it is time to discuss joint surgery with your physician. This surgery can help reverse mobility and flexibility effects and has worked for many patients.
Establish a support system. Arthritis can be extremely overwhelming, frustrating, and even scary. Talking about the symptoms you are experiencing and asking for advice from friends, family members, and other sufferers can help you feel better. There are also many online support groups where you can find the help and understanding that you need.
Physical Therapist
Employ the services of an experienced physical therapist. A physical therapist can develop workout routines with you that will improve joint flexibly and reduce the pain and inflammation associated with arthritis. To get the best results, follow the physical therapist's recommendations regularly, and get back into your daily routines.
You should exercise if you have arthritis, but you should spread out the exercises into brief sessions. You might not be able to perform as many exercises as most people, but the results are more important than your performance. For best results, try three exercise sessions of 10 minutes each to provide you with a half hour of exercise per day.
It is possible that you could benefit from a professional counselor who is trained with emotional issues involved in arthritis. Dealing with a painful illness can damage your emotional well-being, in addition to damaging your body. A licensed therapist can assist you in coping with and understanding your emotions relating to arthritis in a healthy way.
Carrying a heavy bag around can really have a negative impact on your body. Arthritis in a person's shoulders is actually quite common, especially in women. Toting very large bags can cause pain and swelling in the neck and shoulders. Any bag that you carry should be light and should include only the items you really need. Try not to carry it unless absolutely necessary.
When you suffer from arthritis, you should maintain your weight in order to minimize pain from your joints. Your arthritis might end up worse if you are overweight or just have some extra weight. There are other health benefits associated with managing your weight.
As indicated earlier, arthritis painfully affects many people in their daily lives. Having the right knowledge can help you manage your arthritis more effectively.


text: Sorting type: Most visited Most visited Alphabetical | Pages in category "Kids" Benny Rodriguez Squints Palledorous Johnnie Smalls Tommy Santorelli " " Shut up Porter! " - Philips Goes Angry,When Ham Makes a joke about his sister. Phillips Ryan Retrieved from "http://thesandlot.wikia.com/wiki/Category:Kids?oldid=4073" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Characters The Sandlot 2 Characters The Sandlot Charaters The Sandlot: Heading Home Characters Males Females


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ArtWall "Mermaids" Flat/rolled canvas art by Gustav Klimt can be rolled into a free corrugated kraft tube for convenient storage.
ArtWall "Mermaids" Flat/rolled canvas art comes in a horizontal format and is ideal for any home or office decor. Art has 2" accent borders that make it look attractive.
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text:
How Do I Get Squared Off Edges?
Decorating By Chelsea1027 Updated 17 Nov 2009 , 12:56am by sweettooth622
My corners always come out a little rounded on the edges. How can a get a mored squared off edge?
i use setting ganache under my fondant (like Planet Cakes) and it gives a nice crisp base to apply the fondant. Also, using TWO fondant smoothers seems to work wonders for me.
What if you are using butter cream and not fondant?
this video is suuuuper helpful if you are doing a square buttercream cake
What brand of pans are you using? That makes a difference on the sharpness of your edges. Magic Line pans have nice straight sides. Wilton not so much. HTH.
My pans are Wilton Thanks, I'll be on the lookout for better ones next time I plan on doing square. Thank you!


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Jeff Posey (from the album Punchers and Poets and Wildhorse Rdemption)
The third time around for albums show the words and music of Jeff Posey just as charming as his debut (Pocketful). Second album (Mighty Rider) producer Walk Wilkins returns to produce Punchers and Poets and Wildhorse Redemption, the third release from Jeff Posey. Cowboy Americana stages the songs against a western backdrop where memories haunt "Boxcars of Renosa", carefree days of youth play tag in the dust of a rundown house for "Buffalo Nickel", scratchy fiddle and guitar chords find their way through "Indiana" and an assured rhythm keeps company to the lonely lover in "Waiting Up for the Rain". Jeff 'Wildhorse' Posey lends his nickname and demeanor to the four-legged freedom that gallops through "Wildhorse Redemption", slides on strings to speak a tale in the glow of "Pawn Shop Neon", tells the hill country story of "The Lady Llano and Cowboy Kerouac" on a prancing beat, and sings a song for 'dreamers and leavers' as he dances with the wild spirit in "Romancing the Road".
Listen and buy the music of Jeff Posey from his website


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Tumor necrosis factor-α (TNF-α) is a potent inducer of insulin resistance, and increased TNF-α expression is associated with impaired glucose disposal. Although insulin is the primary regulator of glucose transport in adipose, endothelin-1, a vasoconstrictor peptide that signals through the heterotrimeric G proteins Gαq/11, potently stimulates glucose uptake in 3T3-L1 adipocytes by a mechanism independent of phosphatidylinositol (PI) 3-kinase. Here, we report that exposure of 3T3-L1 adipocytes to TNF-α for 48 h dose-dependently decreased endothelin-1-stimulated glucose uptake and translocation of GLUT4 to the plasma membrane. TNF-α exposure had no effect on endothelin-1 receptor number at the cell surface. In contrast, TNF-α treatment reduced the quantity of Gαq/11 and proline-rich tyrosine kinase 2 (PYK2) and decreased endothelin-1-stimulated PYK2-Tyr402 tyrosine phosphorylation. Taken together, these results suggest that TNF-α-induced desensitization of endothelin-1-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes is due, at least in part, to a decreased expression of Gαq/11, leading to a suppression in tyrosine phosphorylation of PYK2.
- adipocytes
- glucose homeostasis
- G protein-coupled receptors
- Gαq/11
- proline-rich tyrosine kinase 2
although insulin is the primary regulator of glucose uptake in muscle and adipose tissue, endothelin-1, a vasoconstrictor peptide secreted primarily by endothelial cells (29), is also a potent stimulator of GLUT4 translocation and glucose transport in 3T3-L1 adipocytes (14, 22, 36). In adipocytes, endothelin-1 action is mediated by binding to the ETA receptor, a G protein-coupled receptor expressed in many tissues and cultured cells including 3T3-L1 adipocytes (4, 36). Although the signal transduction cascade leading from the ETA receptor to the activation of glucose transport is not completely understood, work from a number of laboratories has identified a critical role for Gαq/11 in the endothelin-1 pathway (14, 17, 22). Gαq/11-stimulated glucose transport is independent of phosphatidylinositol (PI) 3-kinase (3, 18, 35, 36). Instead, activation of ETA receptor or Gαq/11 stimulates GLUT4 translocation via a mechanism that requires tyrosine phosphorylation (6, 36) and involves ADP ribosylation factor-6, as well as F-actin polymerization (3, 19). Endothelin-1/Gαq/11 activates the src family tyrosine kinase Yes (13), as well as proline-rich tyrosine kinase 2 (PYK2) (22), in 3T3-L1 adipocytes. Dominant inhibitory constructs of PYK2 prevent endothelin-1 stimulation of F-actin polymerization and GLUT4 translocation (22).
Tumor necrosis factor-α (TNF-α), a cytokine secreted by activated macrophages as well as adipose tissue, is an important mediator of insulin resistance (11). TNF-α production by adipose tissue increases during the development of insulin resistance and obesity in animal models (11), and adipose tissue is one of the major and immediate targets of TNF-α (26). Although it is clear that TNF-α interferes with glucose homeostasis and lipid metabolism, contributing to the development of insulin resistance in obesity and type 2 diabetes (11), its mechanism of action is not completely understood. Exposure to TNF-α leads to serine phosphorylation of insulin receptor substrate (IRS)-1, which inhibits the tyrosine kinase activity of the β-subunit of the insulin receptor and impairs insulin-stimulated signal transduction (12, 27). Increasing evidence suggests that serine/threonine kinases, including protein kinase C and p42/44 MAP kinases, mediate this inhibitory effect of TNF-α on insulin signaling (8, 20). Additional mechanisms of TNF-α-induced insulin resistance may include decreased GLUT4 expression (30), impaired myoinositol incorporation, and Ca2+ mobilization (37), as well as the induction of specific proteins by TNF-α such as suppressors of cytokine signaling-1, -3, and -6, which can negatively modulate insulin signaling (7, 21).
The aim of this study was to determine whether chronic TNF-α exposure inhibits endothelin-1-stimulated glucose transport and to define the components of the endothelin-1-signaling pathway involved in the response to TNF-α exposure. We found that chronic exposure of 3T3-L1 adipocytes to TNF-α decreased endothelin-1-stimulated glucose uptake. TNF-α exposure decreased the expression of Gαq/11 and PYK2, resulting in a reduced endothelin-1-stimulated PYK2-Tyr402 tyrosine phosphorylation and a subsequent decrease in glucose uptake. These results suggest that impaired endothelin-1 signaling may contribute to the development of impaired glucose homeostasis after chronic exposure to TNF-α.
Materials. The murine 3T3-L1 fibroblast cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cell culture reagents were purchased from GIBCO (Grand Island, NY). Antibodies were obtained from the following sources: rabbit polyclonal anti-Gαq/11 (carboxy-terminal) (Calbiochem, La Jolla, CA), rabbit polyclonal anti-GLUT4 (Biogenesis, Sandown, NH), rabbit polyclonal anti-GLUT1 (Chemicon International, Temecula, CA), anti-PYK2 (BD Transduction Labs, San Diego, CA, and Santa Cruz Biotechnology, Santa Cruz, CA), phosphospecific PYK2 antibodies (Biosource International, Camarillo, CA), and anti-cmyc (9E10; Developmental Hybridoma Studies Bank). Goat anti-rabbit and anti-mouse IgG coupled to horseradish peroxidase (HRP) were from Roche (Indianapolis, IN). 2-Deoxy-[3H]glucose and 125I-labeled endothelin-1 were from Amersham (Arlington Heights, IL). Porcine insulin and endothelin-1 were purchased from Sigma (St. Louis, MO). The c-myc-GLUT4 expression plasmid, containing a 14-amino acid sequence of the human c-myc epitope inserted into the first exofacial loop of GLUT4 (16), was from Drs. G. Hermann and R. Kelly (University of California at San Fransisco). Endotoxin-free plasmid kits were from Qiagen (Valencia, CA).
Cell culture and treatments. 3T3-L1 fibroblasts were cultured in Dulbecco's modified Eagle's medium (high glucose) with 10% fetal bovine serum (DMEM-FBS) at 37°C in a 10% CO2 atmosphere for 4 days. 3T3-L1 cells were then cultured in DMEM-FBS containing 10 μg/ml insulin, 0.25 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine for 4 days to initiate differentiation. Medium was then changed to DMEM-FBS with insulin alone, cultured for 3 more days, and then changed to DMEM-FBS for 2 or 3 days before treatment. Under these conditions, >95% of the cell population exhibited the morphological characteristics of adipocytes. TNF-α treatments were initiated 9-10 days after the start of differentiation, at which time 3T3-L1 adipocytes were cultured for 48 h with 0-10 ng/ml TNF-α in DMEM-FBS.
Uptake of 2-deoxyglucose. Uptake of 2-deoxy-[3H]glucose was measured in 3T3-L1 adipocytes differentiated in 24-well plates. After culture with or without TNF-α for 48 h, cells were washed once with serum-free DMEM and incubated in 1 ml of serum-free DMEM for 1 h at 37°C. Cells were then washed twice in PBS containing 0.1 mM CaCl2, 0.1 mM MgCl2, and 25 mM HEPES, pH 7.4, and then incubated in the same buffer in the presence or absence of insulin or endothelin-1 for 30 min at 37°C. In some experiments, 100 nM wortmannin was added 15 min before the addition of insulin or endothelin-1. After agonist treatment, uptake of 10 μM 2-deoxy-[3H]glucose was measured over 10 min. Reactions were terminated by rapidly washing the cells twice with ice-cold PBS. Cells were then extracted in 0.1 N KOH, and aliquots from the cell extracts were counted by liquid scintillation and used to determine protein concentration. Nonspecific uptake was measured in the presence of 10 mM phloretin, an inhibitor of equilibrative glucose transport.
Transient transfection of 3T3-L1 adipocytes and c-myc-GLUT4 cell surface assay. 3T3-L1 adipocytes differentiated in six-well plates were transfected with 2 μg of the c-myc-GLUT4 expression vector using TransFast at a 3:1 ratio according to the manufacturer's instructions (Promega, Madison, WI) for 1 h. The transfection reagent was then removed, and cells were cultured in 2 ml DMEM-FBS for 2 h. Cells were then washed with 1 ml of warm PBS, incubated with 2 mg/ml collagenase and 0.25% trypsin for 5 min to detach cells, and then replated in 96-well plates at 33,000 cells/well. Cells were allowed to attach for 2 h before treatment with or without TNF-α for 48 h.
After 48-h treatment with TNF-α, cells were serum starved for 1 h and then stimulated with or without 100 nM insulin or 10 nM endothelin-1 for 30 min. Cell surface-accessible c-myc-GLUT4 were measured using a cell surface ELISA, as described by Wang et al. (34). Briefly, cells were fixed in 1% paraformaldehyde for 30 min and washed twice for 1 min in 25 mM glycine and twice in PBS. Wells were blocked for 90 min in PBS containing 2% BSA and 1% fish gelatin. Cells were then incubated for 2-3 h with antibody to c-myc (9E10, 2.4 μg/ml) in blocking buffer, washed, and incubated for 1 h in anti-mouse IgG HRP (1:1,000). Cells were then washed and incubated with peroxidase substrate (Sigma) for 30 min, and the optical density at 450 μm was measured. Nonspecific binding was measured in wells incubated without primary antibody.
Isolation of total membranes and subcellular fractions. 3T3-L1 adipocytes differentiated in 100-mm plates were cultured with 0 or 10 ng/ml TNF-α for 48 h. The cells were then serum starved for 1 h and then stimulated with or without 10 nM insulin or endothelin-1 for 30 min. Reactions were terminated by washing the 3T3-L1 adipocytes with ice-cold PBS. Plasma membrane fractions were then isolated as described previously (33) with slight modifications. Briefly, cells were homogenized in a buffer containing 20 mM Tris, pH 7.4, 1 mM EDTA, 255 mM sucrose, and protease inhibitors (Complete; Boehringer Mannheim). After a 20-min centrifugation at 16,000 g, the pellet was resuspended in the same buffer, layered on a 1.12 M sucrose pad, and centrifuged at 100,000 g for 70 min. The interface was then collected and centrifuged at 16,000 g for 15 min to pull down the plasma membrane fraction. In some experiments, total membranes were isolated by centrifuging the homogenate at 200,000 g for 60 min. Equal amounts of proteins (25-50 μg/lane) were solubilized for 15 min at 37°C in Laemmli sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Western blotting was carried out as described previously (23).
PYK2 tyrosine phosphorylation. After TNF-α treatments, 3T3-L1 adipocytes differentiated in six-well plates were stimulated with 10 nM endothelin-1 for 2 or 5 min, washed twice with ice-cold PBS, and then solubilized in 25 mM HEPES, pH 7.4, 1% Nonidet P-40, 5 mM NaCl, 2% glycerol, 5 mM sodium fluoride, 1 mM EDTA, 1 mM sodium vanadate, 1 mM sodium pyrophosphate, and protease inhibitors for 20 min on ice and sheared four times through a 22-gauge needle. The lysates were centrifuged at 12,000 g for 15 min to remove insoluble material. Samples (60 μg of proteins) were then boiled in Laemmli buffer for 5 min and resolved by SDS-polyacrylamide gel electrophoresis.
Competitive 125I-endothelin-1 binding. 3T3-L1 adipocytes differentiated in 24-well plates were cultured with or without 10 ng/ml TNF-α for 48 h. Specific 125I-endothelin-1 binding was determined as described previously, with slight modifications (36). Briefly, the cells were placed on ice, washed, and preincubated in binding buffer (Hanks' buffered saline containing 25 mM HEPES, pH 7.4, 0.2% BSA and 0.1% glucose) with 20 pM 125I-endothelin-1 with increasing concentrations of unlabeled endothelin-1 for 3 h at 4°C. 3T3-L1 adipocytes were then washed twice with ice-cold PBS to remove unbound radioligand and solubilized in 1 N NaOH. Bound 125I-endothelin-1 was then counted by γ-spectrometry. Nonspecific binding was determined in the presence of 1 μM endothelin-1.
Statistical analysis. Data are presented as means ± SE and are compared by Student's t-test or analysis of variance. Analysis of variance was carried out using the general linear model procedure on SAS for personal computers. Differences between treatment groups were then determined by least square means analysis. All data were tested for normal distribution.
TNF-α decreases endothelin-1-stimulated 2-deoxyglucose uptake in 3T3-L1 adipocytes. Although many studies have demonstrated that TNF-α exposure decreases insulin-stimulated glucose transport (11), here we investigated whether chronic TNF-α interferes with endothelin-1-stimulated glucose uptake in 3T3-L1 adipocytes. In control 3T3-L1 adipocytes, endothelin-1 increased 2-deoxyglucose uptake 4.4-fold over basal (Fig. 1) compared with a 7.4-fold increase with insulin. Culture of 3T3-L1 adipocytes with 2, 5, or 10 ng/ml TNF-α for 48 h dose-dependently decreased both insulin- and endothelin-1-stimulated 2-deoxyglucose uptake (Fig. 1).
GLUT4 transporter quantity can be rate limiting for glucose uptake in adipocytes (31). We first asked whether TNF-α supressed endothelin-1-stimulated glucose uptake by decreasing GLUT4 expression. However, chronic exposure of 3T3-L1 adipocytes to 10 ng/ml TNF-α for 48 h had no effect on total GLUT4 quantity (Fig. 2A). 3T3-L1 adipocytes also express GLUT1 transporter, and several reports have shown that TNF-α and other inflammatory cytokines increase GLUT1 expression (5). After treatment of 3T3-L1 adipocytes with 10 ng/ml TNF-α for 48 h, total GLUT1 quantity was increased 2.6-fold over control cells (Fig. 2A).
Endothelin-1 stimulates glucose uptake by promoting GLUT4 vesicle translocation to the plasma membrane. Therefore, we investigated whether the decrease in endothelin-1-stimulated glucose uptake after TNF-α treatment was associated with impaired GLUT4 translocation. In control cells, stimulation of 3T3-L1 adipocytes with 10 nM insulin or endothelin-1 increased GLUT4 protein at the plasma membrane 1.9- and 1.6-fold, respectively (Fig. 2B). However, after chronic exposure of 3T3-L1 adipocytes to 10 ng/ml TNF-α, GLUT4 translocation to the plasma membrane in response to insulin or endothelin-1 stimulation was reduced by 30-40% (Fig. 2B). GLUT1 translocation to the plasma membrane can also be increased by insulin in 3T3 adipocytes and contribute to insulin-stimulated glucose uptake (1). In control 3T3-L1 adipocytes, insulin increased GLUT1 quantity at the plasma membrane 1.5-fold, whereas endothelin-1 had no effect on GLUT1 translocation. Chronic treatment with TNF-α increased GLUT1 quantity at the plasma membrane at baseline, and insulin no longer stimulated GLUT1 translocation to the plasma membrane (Fig. 2B).
Chronic TNF-α exposure also decreased endothelin-1-dependent translocation of c-myc-tagged GLUT4 to the cell surface. The c-myc tag is inserted into the first exofacial loop of the GLUT4 peptide (16); immunological detection of the c-myc epitope at the cell surface is a measure of GLUT4 translocation (34). Stimulation of 3T3-L1 adipocytes with 100 nM insulin or 10 nM endothelin-1 increased surface-accessible c-myc-GLUT4 in control cells 2.6- and 3.3-fold, respectively (Fig. 2C). Chronic treatment of 3T3-L1 adipocytes with 10 ng/ml TNF-α decreased surface-accessible c-myc-GLUT4 in response to both insulin and endothelin-1 stimulation (Fig. 2C), consistent with the TNF-α-induced decrease in endothelin-1-stimulated 2-deoxyglucose uptake and GLUT4 translocation.
Chronic exposure to TNF-α did not change the ETA receptor number or affinity in 3T3-L1 adipocytes. Endothelin-1 stimulates GLUT4 translocation and glucose transport via the ETA receptor, which is the only endothelin-1 receptor isoform expressed in 3T3-L1 adipocytes (14, 35, 36). Therefore, we next asked whether suppression of endothelin-1-stimulated glucose uptake in 3T3-L1 adipocytes in response to TNF-α was due to a decrease in the number of binding sites for endothelin-1 at the cell surface. Competition binding experiments were carried out to determine the levels of ETA receptor density after treatment of 3T3-L1 adipocytes for 48 h with or without 10 ng/ml TNF-α. Neither the Bmax (99.65 ± 0.08% of control) nor the Kd (3.47 ± 0.46 nM in control cells; 2.57 ± 0.22 nM in TNF-α-treated cells) for 125I-endothelin-1 binding was affected by chronic exposure of 3T3-L1 adipocytes to TNF-α (Fig. 3).
Endothelin-1 stimulation of glucose uptake in 3T3-L1 adipocytes is independent of PI 3-kinase. It is well established that insulin stimulation of GLUT4 translocation and glucose transport requires the activation of PI 3-kinase. TNF-α impairs insulin-stimulated glucose transport by inducing a serine phosphorylation of IRS-1, which impairs the ability of insulin to activate PI 3-kinase (12, 27). Endothelin-1-stimulated glucose uptake is independent of PI 3-kinase (17, 36). However, because one group (14) has reported that PI 3-kinase activity is required for endothelin-1 stimulation of glucose transport, we also examined whether PI 3-kinase is involved in the endothelin-1 stimulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes. Pretreatment of 3T3-L1 adipocytes with 100 nM wortmannin for 15 min reduced insulin-stimulated 2-deoxyglucose uptake to basal levels (Fig. 4). In contrast, wortmannin had no effect on endothelin-1-stimulated 2-deoxyglucose uptake in 3T3-L1 adipocytes, consistent with other reports (17, 36) demonstrating that endothelin-1-stimulated glucose uptake in 3T3-L1 adipocytes is PI 3-kinase independent. These data suggest that the mechanisms by which TNF-α desensitizes endothelin-1-stimulated glucose uptake are independent of the effects of TNF-α on the IRS-1/PI 3-kinase pathway.
ETA receptor couples to the Gαq/11 family of heterotrimeric G proteins (4). Gαq/11 is a necessary signaling intermediate in endothelin-1-induced GLUT4 translocation and glucose transport (3, 14). Because endothelin-1-stimulated glucose transport is independent of PI 3-kinase, we postulated that TNF-α-induced desensitization of endothelin-1-stimulated glucose uptake might result from decreased expression of Gαq/11. 3T3-L1 adipocytes were cultured with or without TNF-α for 48 h, and Gαq/11 quantity was meaured by Western blot analysis. 3T3-L1 adipocytes expressed both Gαq and Gα11 polypeptides (Fig. 5). Chronic exposure of 3T3-L1 adipocytes to 2 or 10 ng/ml TNF-α decreased Gαq and Gα11 quantity by 50 and 70%, respectively (Fig. 5).
TNF-α reduced total PYK2 and endothelin-1-stimulated PYK2-Tyr402 phosphorylation in 3T3-L1 adipocytes. Tyrosine phosphorylation is required for endothelin-1 stimulation of GLUT4 translocation and glucose transport. PYK2, a cytoplasmic tyrosine kinase known to be required for endothelin-1-stimulated glucose uptake (22), is tyrosine phosphorylated in response to endothelin-1, but not insulin, stimulation. Because chronic exposure of 3T3-L1 adipocytes to TNF-α decreased Gαq/11 expression, we hypothesized that endothelin-1 stimulation of PYK2 tyrosine phosphorylation would be decreased in TNF-α-treated adipocytes compared with controls. Although PYK2 can be tyrosine phosphorylated on four residues, activation of PYK2 is dependent on tyrosine phosphorylation on Tyr402 (2). Using a PYK2-Tyr402-specific antibody, we found that endothelin-1 increased PYK2-Tyr402 tyrosine phosphorylation in control cells, with a maximum phosphorylation observed after 5-min stimulation with endothelin-1 (data not shown). Chronic exposure of 3T3-L1 adipocytes to 10 ng/ml TNF-α decreased total PYK2 expression by 50% (Fig. 6). Endothelin-1 stimulated the phosphorylation of PYK2-Tyr402 1.6-fold over baseline in control adipocytes (Fig. 6). However, after chronic exposure to TNF-α, endothelin-1-stimulated tyrosine phosphorylation of PYK2-Tyr402 was decreased to only 60-70% of the phosphorylation observed in controls (Fig. 6).
The key finding of the present investigation is the observation that chronic treatment of 3T3-L1 adipocytes with TNF-α induced a marked inhibition of endothelin-1-stimulated glucose uptake. Although TNF-α has potent inhibitory effects on insulin signaling, the extent to which this important cytokine can affect endothelin-1-stimulated glucose uptake in adipocytes has not been previously investigated. Here, we report that TNFα exposure decreases endothelin-1-stimulated glucose uptake by impairing GLUT4 translocation to the plasma membrane. Although the signal transduction pathways leading from endothelin-1 receptor activation to GLUT4 translocation are not completely understood, it is clear that Gαq/11 and PYK2 are critical intermediates in this process (3, 14, 22). After chronic exposure to TNF-α, the expression of both Gαq/11 and PYK2 was decreased in 3T3-L1 adipocytes. This reduction was associated with an impaired ability of endothelin-1 to stimulate the phosphorylation of PYK2-Tyr402, demonstrating that TNF-α suppresses endothelin-1-stimulated glucose uptake by interfering with the signal transduction cascade required to activate GLUT4 translocation in 3T3-L1 adipocytes.
Prolonged exposure to TNF-α, especially at periods longer than 72 h, induces dedifferentiation of fully differentiated adipocytes, including a decrease in GLUT4 expression (8). Several reports have demonstrated that TNF-α-induced insulin resistance is associated with decreased GLUT4 expression (30, 32). However, under the conditions of TNF-α exposure used in our model system, no changes in the 3T3-L1 adipocyte morphology and lipid content were observed (data not shown). Furthermore, suppression of endothelin-1-stimulated glucose transport by TNF-α was not associated with a decrease in total GLUT4 protein quantity. Instead, decreased insulin and endothelin-1-stimulated glucose uptake after TNF-α exposure was associated with impaired GLUT4 translocation to the plasma membrane. Although chronic TNF-α exposure for 48 h had no effect on GLUT4 expression, the quantity of GLUT1 was increased. Cytokines, including TNF-α and inter-leukin-1, have been shown to upregulate GLUT1 and increase basal glucose uptake through GLUT1 (5). However, this increase in GLUT1 quantity in response to TNF-α exposure was not sufficient to compensate for the loss of insulin- and endothelin-1-stimulated glucose uptake.
3T3-L1 adipocytes contain a single class of endothelin-1-binding sites identified as the ETA receptor sub-type (4, 36). One possible consequence of chronic exposure to TNF-α could be a decrease in endothelin-1 receptor number and affinity leading to less endothelin-1 stimulation of glucose transport. However, competition binding experiments showed that the Bmax and Kd for binding of endothelin-1 to the ETA receptor were not changed by chronic exposure of 3T3-L1 adipocytes to TNF-α, suggesting that TNF-α exposure affected endothelin-1-dependent signaling downstream of the ETA receptor.
Insulin and endothelin-1 have additive effects on glucose uptake in adipocytes (25, 36). Endothelin-1-stimulated glucose uptake in 3T3-L1 adipocytes is independent of PI 3-kinase. Instead, numerous studies have demonstrated a critical role of Gαq/11 proteins in the endothelin-1-stimulated (14), as well as insulin-stimulated (15), GLUT4 vesicle translocation and glucose transport (17). Overexpression of constitutively active forms of Gαq (Q209L) or Gα11 (Q209L) in 3T3-L1 adipocytes stimulates F-actin polymerization, GLUT4 translocation, and glucose uptake (3, 18). Conversely, microinjection of RGS2 (regulator of G protein signaling 2), a protein that inhibits Gαq/11 activity, or microinjection of antibodies against Gαq/11 inhibits GLUT4 translocation (14). Prolonged exposure to TNF-α has been reported to modulate the expression of heterotrimeric G proteins in different cell types (9, 10, 24, 28). In the present study, we show that chronic exposure of 3T3-L1 adipocytes to TNF-α decreased Gαq/11 expression. This decrease in Gαq/11 quantity likely contributes to the TNF-α desensitization of endothelin-1-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. Moreover, because Gαq/11 has also been implicated in insulin-stimulated GLUT4 translocation, TNF-α-induced decrease in Gαq/11 expression may also contribute, in conjunction with impaired insulin receptor signaling (11), to the effects of TNF-α on insulin-stimulated glucose uptake in 3T3-L1 adipocytes.
Insulin and endothelin-1/Gαq/11 utilize distinct signaling pathways to stimulate GLUT4 translocation and F-actin polymerization (22). PYK2, a Ca2+-sensitive protein tyrosine kinase, is a downstream effector in the endothelin-1/Gαq/11, but not the insulin, stimulation of GLUT4 vesicle translocation to the plasma membrane (22). Dominant inhibitory constructs of PYK2 (CADTK-related nonkinase, CRNK) inhibited endothelin-1 but not insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes (22). Because TNF-α-induced desensitization of insulin-stimulated glucose uptake is primarily due to impaired insulin signaling via the PI 3-kinase pathway (11), we hypothesized that the effects of TNF-α on endothelin-1-stimulated glucose uptake would reveal an additional signaling pathway susceptible to regulation by TNF-α in 3T3-L1 adipocytes. Here, we show that endothelin-1 stimulates the phosphorylation of PYK2-Tyr402 in 3T3-L1 adipocytes. Importantly, when 3T3-L1 adipocytes were exposed to 10 ng/ml TNF-α for 48 h, expression of total PYK2 was decreased and endothelin-1 stimulation of PYK2-Tyr402 phosphorylation was impaired. These data identify PYK2 expression and phosphorylation as new targets for regulation by TNF-α and suggest that TNF-α-induced decreases in Gαq/11-mediated signaling, in combination with decreased PYK2 protein quantity, together contribute to TNF-α-induced desensitization of endothelin-1-stimulated glucose transport.
This work was supported by US Public Health Service National Institute on Alcohol Abuse and Alcoholism Grant R01 AA-11876. The antibody to c-myc (9E10), developed by J. M. Bishop, was from the Developmental Studies Hybridoma Bank, maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA) under contract NO1-HD-7-3263.
We thank Gary Herman and Regis Kelly (UCSF) for the c-myc GLUT 4 expression vector.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2003 by American Physiological Society


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Let it rip, Potato Chip! Australia's favourite snacks
When it comes to the nation's most popular snacks, it seems Australians' tastes tend more towards savoury than sweet. In an average seven-day period, 41% of us snack on potato chips, 37% snack on nuts, and 32% snack on savoury biscuits/crackers. But while these salty treats are the nation's undisputed favourites, sweet snacks outnumber them when we consider the top 15 snacks enjoyed by Aussies 14+.
The latest findings from Roy Morgan Research show that among the 15 snacks most commonly eaten by Australians in an average week, five are savoury, nine are sweet and one (plain/natural yoghurt) is 'neutral'.
Snack attack: Australia's most popular snacks
Source: Roy Morgan Single Source (Australia), July 2013 – June 2014 (n=16,809)
Sugar, spice and all things nice?
Women tend to be more likely than men to opt for snacks that are generally considered to be healthy, such as nuts (39% vs 35%), savoury biscuits/crackers (35% vs 28%), natural/plain yoghurt (32% vs 20%) and health/muesli/fruit bars (18% vs 15%).
Nutritional value and/or calories appear to be of less concern for men, who are more likely than women to snack on potato chips (44% vs 38%) and corn chips (19% vs 15%) in an average seven days.
Ice cream is the great leveller, enjoyed by similar proportions of women and men, whether it's from a tub (27% of women vs 28% of men) or on a stick (19% of both).
Older Aussies: nuts for nuts
Age is also a determining factor in Australians' snacking habits. For example, in any given seven-day period, 60% of teenagers under 18 and 50% of 18-24 year-olds snack on potato chips — compared with 34% of 50-64 year-olds and 23% of those aged 65+.
What's more, younger Australians (and those up to 49 years) are more likely than the 50-plus brigade to tuck into everything from lollies to chocolate bars, corn chips and health/muesli/fruit bars.
Among the more mature demographics, nuts and savoury biscuits/crackers are more popular than potato chips, with 50-64 year-olds being the age group most likely to snack on nuts in an average seven days and Aussies aged 65+ being the biggest fans of savoury biscuits/crackers.
Angela Smith, Group Account Director, Roy Morgan Research, says:
"With two of every five Australians 14+ saying they 'tend to snack throughout the day', the snack market in this country is huge. While potato chips remain the nation's undisputed favourite snack, their popularity varies between men and women and different age groups. Indeed, this is true of most snacks.
"But age and gender are just two factors that influence a person's snacking habits: attitudes towards food are obviously another. For instance, more than three of every five Australians say they 'prefer to eat healthy snacks' and nearly the same amount restrict how much they eat of fattening foods. Predictably, these people are more likely than others to opt for healthier snacks such as nuts and muesli bars.
"Add sweet tooths and savoury cravings to the equation, and the snack food market gains another degree of complexity. This is why an in-depth understanding of Australian snack consumers, and their diverse habits, attitudes and preferences, is so vital for snack-food brands to ensure they catch the attention of their target market."
Contact Profile
Roy Morgan Research
Roy Morgan Research is Australia's best known and longest established market research and public opinion survey company. Roy Morgan Single Source is thorough, accurate, and provides comprehensive, directly applicable information about current and future customers. It is unique in that it directs all the questions to each individual from a base survey sample of around 55,000 interviews in Australia and 15,000 interviews in New Zealand annually - the largest Single Source databases in the world. The questions asked relate to lifestyle and attitudes, media consumption habits (including TV, radio, newspapers, magazines, cinema, catalogues, pay TV and the Internet), brand and product usage, purchase intentions, retail visitations, service provider preferences, financial information and recreation and leisure activities. This lead product is supported by a nationally networked, consultancy-orientated market research capability.
Samantha Wilson
W: www.roymorgan.com


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"Davidson" Submits:
One might think that with the thousands of very bright people, 100s of thousands of MBA decreed over the decades and the number of Nobel Prizes conferred that someone somewhere would have by this time identified what is the "Fair Market Price" of investment markets. The facts revealed by historical study reveal that many opinions are present at all points during an investment cycle and that many accept the concepts of "The Invisible Hand" and "The Efficient Market Theory". Each in its own way declares that "Fair" market prices are established by the confluence of buyers and sellers in the prices which we see every day.
For those of you who have received my notes for some time, you know that I disagree with the concept of markets setting prices at "fair" levels. Markets are human systems! Human systems invariably imply higher social value to some items over others and general social consensus confirms this to be true. In the financial markets securities can be priced well away from their measurable long term returns and often are, due to "Market Psychology". We have actually seen this fairly frequently since 1995 since Hedge Funds emerged to become a significant influence of market prices. In the chart below, SP500 Index (SPDR S&P 500 ETF Trust (NYSEARCA:SPY)) vs. S &P 500 (.INX) Intrinsic Value Index, I show the SP500 against an index which I created which I think provides a reasonable measure of market "fair" pricing which I call the SP500 Intrinsic Value Index. Think of this index as representing the market's "Fair Price" vs. "Market Psychology" Prices which are represented by the SP500. This benchmark comes from the "Prevailing Rate"
Since 1995, Hedge Funds have become a dominant influence in market pricing. We know this by reading the literature. We have also have had one investment "Bubble" after another with the Internet Bubble in 2000, the Housing Bubble (SPDR S&P Homebuilders (ETF) (NYSEARCA:XHB)) in 2005-6, the Peak Oil Bubble in 2007-8 (United States Oil Fund LP (ETF) (NYSEARCA:USO)), the Fear of Inflation and Collapse of the US$ Bubble in 2011 (Gold (SPDR Gold Trust (ETF) (NYSEARCA:GLD)) peaked $1,867.60 Sept 9, 2011), Fear of Euro Collapse 2012(10yr Treas Rates bottomed 1.394% July 2012) and there have been mini-"bubbles" as cash has rapidly shifted from one situation to another. This type of behavior is actually not different from the past, but it is occurring much more quickly. Along the way any sense of "fair" pricing of tends to evaporate as investors seek to avoid losses and reap gains by following what "the market thinks". Those who believe that the "market knows all" and chase each trend include most of those people on CNBC with "something significant to say" to say every day.
Prices often do not represent the business returns of the underlying businesses. Take Tesla Motors Inc (NASDAQ:TSLA) for example. Tesla Motors Inc (NASDAQ:TSLA) is currently priced at ~15bil, but has less than 1bil of Revenue (a Price to Sales factor of ~15) and the TTM (Trailing 12mos) Earnings Per Share is reported as a loss of ~$2.70/shr for investors. This investment is trading very much like the Internet stocks of the late 1990s. Tesla does not have as yet a business return one can measure with which to predict if today's price is worth buying in comparison to all of the opportunities available. This is a news driven investment as Tesla has had quite of bit of press with Founder/CEO Elon Musk being very smart and always in the news. I have no idea if Tesla will eventually prove to be an investment which actually trades on a measurable business return, but history indicates that it will, eventually. When it does, it will have to do much better than Ford Motor Company (NYSE:F) which sells at ~0.5 x Revenue of 130bil and Toyota Motor Corporation (NYSE:TM) which sells at ~0.9 x Revenue of 220bil. Ford and Toyota have measurable business returns and are deemed, considering how they are currently managed, to be very good companies. My net/net is that Tesla Motors Inc (NASDAQ:TSLA) even with the brilliance of Musk looks to be in a significant "bubble" to me. These types of events occur primarily because, in my observation, investors do not have a historical pricing benchmark and assume that the market's "Invisible Hand" provides "fair" pricing. What is actually happening is that investors are amplifying each other's investment activity. Believing that some one knows more than they do, these investors pile into the trend of the moment driving prices to an extreme. There is a term given to these types of investors which many wear proudly; they are called Momentum Investors. (I do not recommend any of the companies discussed.)
To counter this one begins by constructing a valuation benchmark using economics. The difficulty is developing a benchmark is that economic conditions vary across the business cycle and even across wide swaths of our economic history. But, with study, one can come to the conclusion that in order for stocks to even exist, they must provide a return higher than the general economy or otherwise investors would not have interest. This means that the US long term GDP has the means to be our valuation benchmark. If the measurable returns of one company are significantly better than the general economy and other companies over time, then the stock of this company should sell significantly higher than other stocks and in proportion to its excess performance vs. GDP. It turns out that when one does all the work, of the components of GDP, Real GDP has long term predictability while inflation does not. What one does then is to use the long term trend (~25yr trend RGDP) of Real GDP and add back into it the best measure of inflation on a 12mo basis one can identify. This is where the "Prevailing Rate" comes from.
The "Prevailing Rate" is used to capitalize the reported Net Income of the SP500. But, I do not use today's reported numbers which are high relative to the longer term mean, I actually use the SP500 Mean Earnings. The reason for doing this came to me a number of years ago as I was reading the various announcements of known Value Investors buying into severe market downturns. Warren Buffett and other true Value Investors when they make an investment are buying an earnings stream which is at least 5yrs-10yrs in the future. Good Value Investors are buying stocks for their representative company's future earnings. They are NOT buying these issues based on currently reported earnings disasters.
Value Investors are disciplined buyers of business returns. They price a company based on its long term earnings stream using the context of the expected GDP trend. The "Prevailing Rate" represents that context as best I think it can be estimated. The SP500 Long Term1, 2 - View Full Page


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Discussion in 'Hardscaping' started by Marek, Mar 1, 2006.
Whats the cost difference between a paver patio and a concrete patio.
This is a great question, I have been wondering the same thing. I have been planning on doing my front walk way, would love to do a paver walkway, but I was not sure what the savings would be if I went the concrete route.
Up here, concrete can be done for $ 2 to 4 per square foot. Pavers go from $10 to ???
Not sure the cost of concrete around here, only sure thing is it is considerably cheaper than laying pavers. I have one customer I'm in the process of trying to sell now that is leaning to a stamped concrete patio because of the cost issue (I believe about one third the cost of pavers). Around here I get $15 on up per square foot for pavers.
I was talking to a builder and I was asking him about stamped concrete. He said it goes for around $12/sq. ft. and up.
Yep, pavers - $15/sf+
PIP concrete is around $5/sq.ft. in this area, stamped concrete hovers close to $10/sq.ft. And paver are from about $9 and up.
We won't pour for less than $9.00 per SF. Pavers can go for as little as 10, and up to $15.00 per SF. In many cases, Stamped sells for more than pavers.
Also, color dictates price. Some colors on the Davis chart can add $100.00 per yard to the mud cost.
Stamped concrete $10 all day every day up here....pavers $14-$15
Not sure about concrete cost. We install pavers from $10-$15 sq. ft. We tell are customers when they ask about concrete the only guarantee that you get with concrete is that it will get hard and crack. Which is the truth. So when you have to replace the concrete, pavers would have been cheaper in the long run.


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The effects of management of genetically modified herbicide–tolerant (GMHT) crops on adjacent field margins were assessed for 59 maize, 66 beet and 67 spring oilseed rape sites. Fields were split into halves, one being sown with a GMHT crop and the other with the equivalent conventional non–GMHT crop. Margin vegetation was recorded in three components of the field margins. Most differences were in the tilled area, with fewer smaller effects mirroring them in the verge and boundary. In spring oilseed rape fields, the cover, flowering and seeding of plants were 25%, 44% and 39% lower, respectively, in the GMHT uncropped tilled margins. Similarly, for beet, flowering and seeding were 34% and 39% lower, respectively, in the GMHT margins. For maize, the effect was reversed, with plant cover and flowering 28% and 67% greater, respectively, in the GMHT half. Effects on butterflies mirrored these vegetation effects, with 24% fewer butterflies in margins of GMHT spring oilseed rape. The likely cause is the lower nectar supply in GMHT tilled margins and crop edges. Few large treatment differences were found for bees, gastropods or other invertebrates. Scorching of vegetation by herbicide–spray drift was on average 1.6% on verges beside conventional crops and 3.7% beside GMHT crops, the difference being significant for all three crops.
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idea of a girl's body shape he really likes is...you hear some guys say they want there girls to be soft not skinny and then there's the media or guys gawking at girls on the magazines covers. as a girl I'm really curious as to what you guys really want in a girl physically and realistically (be honest) so I thought it would be interesting if some guys would find pics to post on what is appealing...really curious stomach wise. maybe some girls are too? haha
Most Helpful Guy
Personally all the girl's body shapes are attractive from the skinny body type to the curvy body type and a woman is beautiful with her body. Confidence is definitely attractive so you should love your body and the guy will like it too. I believe guys in general place more emphasis on a girl's personality than their body. A girl's personality is what makes her attractive because a guy would love to hang with and have fun with a girl that has a great personality.00|00|0


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Contact Info / Websites
Recent Art Reviews
Inktober 2016
Rated 5 / 5 stars
I'm kinda late to comment on this compilation, but you really nailed this Inktober. You have a lot a variety here, sexy girls, animals, backgrounds, humor... All in beautiful inks.
Rated 5 / 5 stars
Would like to see cartoon with this character design. Your cartoon anatomy is great. Hands especially.
IkaroTsubasa responds:
Thanks a lot! :D
Rated 5 / 5 stars
Love how you add the personality to your cartoons. Middle pic is great.
IkaroTsubasa responds:
Thanks! :D


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Hi all,
Simple question, is there a way in Windows to determine what wireless encryption options a wireless card supports? I am working with a laptop that is a few years old and is having trouble connecting to a wireless network. I am guessing it is because the wireless card does not support the type of encryption being used (WPA2).
Thanks for the help.
1 Reply
· · ·
Jun 20, 2012 at 11:42 UTC
If you try to add a wireless network manually I think you can see what is built in:
But I think certain cards can also have other types of encryption added.
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


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Rayden Steele was born in Georgia, but has his roots in Jamaica; this "peach" is about to be eaten on StraightRentBoys.com. Rayden's gay and his diet of Cheetos keeps him young and fit; oh, youth. As Rayden starts to "stretch" out, Eddie calls in straight guy Bruno to help; he'll be "kneeling down" to assist. Shit, Rayden's got a nice cut cock and a full sack.
Working together, the boys seem to have it "well in hand, and mouth;" Rayden spreads his legs, pumps his hips and eagerly moans as he watches Bruno suck. Wanting to get a better feel of Bruno, Rayden's hand begins to wander, which has him edging. Putting lube on Rayden's cock, Bruno pumps away; his partner gasps, gives a, "fuck," and oozes out.
Length: 875 MiB Duration: 16mn 8s 21ms
Video: AVC at 7 325 Kbps, Aspect: 1920 x 1080 (1.778) at 29.970 fps
Audio: AAC at 256 Kbps, Infos: 2 channels, 48.0 KHz
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Recently had my account blocked on Virtual City Casino after wagering the bonus until I could withdraw my winnings. They claim that I have to call the risk management team. every time I phone it's apparently a different country and time of day. Very bad casino, keeping all the money, locking accounts, customer service is a joke (first woman i talked to was laughing at the fact I cant buy people things i promised) Was sent withdrawl confirmation but never contacted regarding this decision. avoid this place. We are going to write letters and keep calling but they just don't care about the consequences. "
Please send your account number to [email protected] so I can look into your issue.
The casino support is located in Australia. Current timezone is +11:00GMT. Risk management department is in the office between 8.30-5.30.
Actually I have managed to find your account.
I have spoken to risk management and they have asked that if you are not able to get ahold of them via phone, you can email them at [email protected]
Any updates from the submitter of this complaint?
What happened here? Any updates from the player?
There's no need to affect casino's reputation with posting a complaint which closes due to inactivity of the submitter, after tho chances given to respond, so this case is solved!
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M.C. Blanchard Judicial Building
190 Governmental Center
Pensacola, FL 32502
Submitting Proposed Orders
Please e-mail ALL proposed orders in Microsoft Word with the case name/style; case number; and title of the proposed order specified in the subject line to: . Other correspondence should be sent to: .
Example - Word Document should be named: Smith 2016 DR 9999
Remember to copy all parties of record in that transmission.
A minimum of one week should be allowed for the processing of all proposed orders. Once entered, the Court will forward all orders to the Escambia County Clerk so that the Clerk's Office may furnish the orders to the parties via e-service.
NOTE: Please continue to file all other pleadings/motions with the Clerk of Court. Courtesy copies are welcomed with the emailed proposed orders; however, any courtesy copies furnished for the Judge should be submitted only as a copy of the document filed with the Clerk of Court which includes the electronic filing number, date and time stamp. Motions received at the judge's office via e-mail, or any other means, are not considered "filed" and will not be processed until properly filed with the Clerk of Court. Any motions/pleadings received without the electronic filing information will be rejected as "not part of the record".
PRO SE - If you are not represented by an attorney, please send your proposed documents to:


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Registration takes less than 1 minute.
The logistics software company will pay $107 million for freight visibility provider MacroPoint, one of the hottest properties in logistics technology.
The Port of Los Angeles is looking to open a container staging facility on a parcel of property right in the middle of Terminal Island, home of the nation's busiest containerized cargo terminal.
In an effort to diversify its cargo operations, the port of Salalah in Oman looking to establish a sugar plant and has begun talks with investors, according to local media source Oman Daily Observer.


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Question: The recent study by Ace Metrix found Papa John s John
The recent study by Ace Metrix found Papa John's John Schnatter and Boston Beer Company's Jim Koch to be the most effective CEO endorsers. Find an example of an advertising message in which each appears and analyze it. Why do you think Schnatter and Koch are such effective endorsers?
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Answer to relevant QuestionsNBA star Kobe Bryant has been able to rebuild his image after charged with sexual assault a number of years ago. Do you agree with the decision by companies and brands such as Turkish Airlines and Vitamin water to use Bryant ...Discuss the three primary source attributes noted by Herbert Kelman and the different processes by which they can influence attitude and/or behavior change. Find an example of an advertisement or other type of promotional ...Why is it important for marketers to set specific objectives for advertising and promotion? What criteria must these objectives meet to be valid?Many advertising creative personnel are opposed to focus groups research as they argue that they may inhibit the creative process. Discuss the problems, as well as the value, of using focus groups to evaluate advertising ...IMC Perspective 8-2 discusses how Siemens developed the "Answers" campaign to increase awareness of the company and create a stronger brand identity. Evaluate the big idea used for the "Answers" campaign and discuss ...
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We designed this Plaza skate park to mimic obstacles found in nearly every city. Plaza Decks with Ledges, Grind Boxes and a Skate Barrier deliver challenges for all levels. Plus, Manual Pad combinations and more Grind Boxes provide spaces for riders—beginners to more advanced—can perfect their tricks and skills.
Component Colors
Product Lines
Project Price Range
*prices vary. For international and exact pricing, please contact your local playground consultant.
Age Ranges
- 5 to 12 Years
- 13+ Years
Design Standards
Min Area Required
100' x 65' (30,48 m x 19,81 m)
Product Lines Featured in this Playground
Ages 5 to 12, Ages 13+
Skatewave® modular skateparks allow riders to enjoy the peak performance and challenge that only comes from precision engineering, innovative products, custom skatepark design and the very best materials. Skatewave is the first system with a totally interchangeable riding surface and totally modular components for endless custom skatepark designs.


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Prince Dorgon is one of two villains of 2012 South Korean action film War of the Arrows is Qing Prince who invaded Korea along with his uncle and general Jyustina whom they closed as father and son . He was cruel and dangerous prince who raped Korean women as slaves and massacred their loved ones. until he chose Choi Ja-in, a recently married woman whose wedding innputed by attack which killed her in laws who happened be foster parents.the men tried off her clothes Ja-in speak Manchu from her late father who surprised and impressed Dorgon who begun lust on her and sent her food. next night Ja-in tried killed Dorgon who tried raped her before she was saved by her brother Choi Nam-yi and her husband Kim Seo-goon and their friends, Nam-yi holding Dorgon as his hostage in front of the guards asking his sister and brother in law to escaped it along with his friends Nam-yi used Dorgons's beers splash into him and used bedsheets it take few hours before Nam-yi used bow to make fire which kills Dorgon who burned to death in front of his bodyguards, his death left Jyustina to get revenge against Choi Nam-yi.
- He was played by Park Ki-woong who played Shunji Kimura
- Unlike in the movie Dorgon survived in the real life and rule Qing under title as Prince