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Enable RADIUS authentication in the LOM GUI
RADIUS (Remote Authentication Dial-In User Service) is a network protocol that allows you to manage remote user authentication, authorization, and auditing.
Configure RADIUS authentication in the LOM GUI
- Log on to the LOM GUI.
- Navigate to Configuration > Radius.
- Select Enable RADIUS.
- Specify the port and IP address of the RADIUS server.
- Specify the secret (password) for the user to access the RADIUS server.
- Click Save.
RADIUS username and password:
The RADIUS server determines the supported characters and lengths. BMC does not limit or filter any character.
On the MPX 8000/8600, MPX 14000, and MPX 25000 series platforms, BMC password length support: 1 character minimum, 16 character maximum.
On the MPX 8900, MPX 15000, and MPX 26000 series platforms, BMC password length: 1 character minimum, 64 character maximum.
RADIUS secret:
BMC valid characters are 0–9, a-z, A-Z,! @, +, -, /,: and _
On the MPX 8000/8600, MPX 14000, and MPX 25000 series platforms, BMC secret length support: 1 character minimum, 31 character maximum.
On the MPX 8900, MPX 15000, and MPX 26000 series platforms, BMC secret length: 1 character minimum, 128 character maximum.
Configure an external computer running Windows or Ubuntu OS to access the LOM using RADIUS' network protocol
RADIUS is an application that runs on an external computer to access the Citrix LOM. Before you run RADIUS, you need to configure the user account and client information.
Perform the following actions:
- Configure a user account in Ubuntu.
- Configure client information in Ubuntu.
- Start the RADIUS server in Ubuntu.
- Add roles in Windows server.
- Add an object – Group.
- Add an object – User.
- Add a network policy.
- Add a vendor specific attribute.
- Configure a RADIUS client.
For more information, see the documentation for Ubuntu.


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Image courtesy of Stuart Miles / FreeDigitalPhotos.net
I know you all love sweepstakes so every Sunday I will be posting a list of live sweepstakes to enter. All official rules for each sweepstakes can be found on their website. Let me know if you win anything! If you know one I am forgetting please feel free to add it in the comments for next week roundup!
Boogie Wipes The #1 Pick Sweepstakes ($50 Amazon) (Pinterest contest)~ Open to US & Canada (excludes Quebec) Ends 3/31/2014 ~ Enter here (a new winner chosen every 3 days)
Purex $1,000 Shopping Spree ~ Open to US ~ Ends 1/31/14 ~ Enter here
Martha Stewart Living Daily Giveaway (daily jewelry giveaway)~ Open to US~ Ends 1/31/14 ~ Enter here daily
Honey Bunches of Oats "Bunch of Beats" Sweepstakes ($500 weekly) ~ Open to US ~ Ends 6/15/14 ~ Enter here weekly
WomanFreebies HP Laptop Sweepstakes ~ Open to US & Canada ~ Ends 2/13/14 ~ Enter here daily
Pepsi/Coin Toss Sweepsakes ~ Open to US (excludes Alaska, Hawaii, Montana, Oregon, and Washington State) ~ Ends 2/3/14 ~ Enter here daily
31 Days of RockaRoo ~ Open to US (excludes RI) ~ Ends 1/31/14 ~ Enter here (winner picked daily)
Snickers Super Bowl Satisfaction Instant Win Game ~ Open to US ~ Ends 2/28/14 ~ Enter here daily
Rogue Stunt Giveaway (3 will win a Nissan Rogues valued at $30,000) ~ Open to US and District of Columbia ~ Ends 2/10/14 ~ Enter here daily

subset: commoncrawl


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The 1941 season was one of the most important transitional years in college football history, even if nobody really knew it at the time. It is the year where Mizzou arguably impacted college football more than it ever had before, and ever would again. It was the year that Don Faurot unveiled the Split-T formation.
He did not do it because he thought it would change football, of course — he did it because he had just lost his star player (Paul Christman) and needed to figure out creative ways to move the ball down the field.
Mizzou had enough potential that it was named Big 6 conference co-favorite.
Missouri, Oklahoma and Nebraska are on a virtual par with the schedule giving the Tigers their edge. They play both of their strongest rivals at home and won the 1939 flag with a similar program.
The fight for fourth place should be every bit as close between Kansas State, Iowa State and Kansas.
Still, Faurot thought he needed something extra.
Let's once again revisit Bob Broeg's Ol' Mizzou: A Story of Missouri Football. A lengthy, phenomenal passage:
As Jim Conzelman and Bud Wilkinson said, the Split-T formation was the greatest offensive contribution in a quarter-century. The quarterback option play, the fascinating keep-or-pitch that distinguished it, has been incorporated into just about every offensive offshoot since then.
What led Faurot to this ingenious juxtaposition of "x's" and "o's"? The thin man was looking for something. Christman gone, he remembered with a shudder the disaster that followed the departure of Jack Frye's passing arm in 1937. Faurot recognized that he had exceptional speed in hip-swiveling 165-pound [Harry] Ice and 180-pound cutback artist [Red] Wade, and an exceptional runner in big 200-pound [Bob] Steuber. Steuber's effectiveness on the end-around had convinced the coach he was a running back, not a receiver.
In 1940 the modernized T-formation had sprung upon the football scene with powerful impact. George Halas with the Chicago Bears and Clark Shaughnessy at Stanford University had refined Ralph Jones' man-in-motion version of the ancient T.
The quarterback no longer squatted behind the center, but merely crouched, hands under the crotch so that he handled on a direct handback, rather than a snap. A halfback went in motion to create better passing situations.
The Bears had slaughtered the Washington Redskins in the National Football League's championship game, 73-0. Stanford had surged from nowhere to the Rose Bowl and defeated Nebraska, 21-13.
Faurot borrowed Nebraska's Rose Bowl film from Biff Jones, investigated, and then made up his mind. He ruled out the man-in-motion because he reasoned that last-second movements by the defensive players made it more difficult for offensive linemen to carry out their blocking assignments. Nor did he like the tight offensive alignment of the regular T, feeling that this cozy, shoulder-to-shoulder offensive line made it easier for defensive linemen, unless blocked thoroughly, to reach out and stop or delay a ballcarrier. Faurot, who liked to spread his line a bit even in the single wing, decided to split his offensive line, leaving a 12-inch space between center and guard, two feet between guard and tackle, and a yard between tackle and end.
Additionally, not figuring on his quarterback to do as much passing as the halfbacks on a run-or-pass option that simulated the cocked-arm threat of a single-wing or short-punt tailback sweeping wide, Faurot tinkered to give his quarterback more mobility and potential as a runner. So he eliminated the quarterback pivot from under center and substituted, instead, a slide that, he felt, would permit more deception and faster handoffs by making the ball exchange to the running back closer to the line on dive plays. Moreover he wanted the quarterback keep-or-give option for a very good reason.
Looking back at the results of his other formations, Faurot had found that his most consistent ground-gainer was a play he had put into the short punt to offset Paul Christman's lack of quickness. To get big Dooz into motion, the coach had devised a play in which the direct snap went not to either of the deep men, the tailback or fullback, but to the closest back. Jim Starmer, the halfback, would reverse into the direction Christman had started. Starmer would either flip a lateral (or pitchout, as it would be called now) to the tailback coming around or fake and go inside end himself. Both plays had averaged well, especially Starmer's fake and keep.
"So I wanted to pressure the defensive end with what amounted to a 2-on-1 break in basketball," said Faurot.
The Split-T could better be called the Sliding-T or, as the coach later suggested, the Missouri-T, or if he had not been so darned modest, the Faurot-T. It succeeded beyond his expectations.
Brawny, brainy Harry Smith, the former Trojan star fresh from a year at guard with the Detroit Lions and only 22 years old, was an English major who never let a simple word stand in the way of the magnificent.
"Don didn't know how volatile the Split-T was," said Smith, years later retired from coaching and well-contented as a physical education professor at Ol' Mizzou. "It was gigantic ... colossus."
In other words, pretty good.
The 1941 season saw the perfect confluence of innovative strategy and improving talent at Mizzou. With Faurot putting his new formation into the heads of a mercilessly fast and physical backfield, the results were staggering.
In 1941, Mizzou averaged fewer than two completions and 40 passing yards per game, but the Tigers also led the country with over 300 per game on the ground. Steuber rushed for 855, Wade 681, and Ice 603. As Broeg put it, theirs was by far the best offense in the midlands. Oh yeah, and their defense allowed only four points per game, three per game after the season opener.
Before we walk through that 1941 season, however, let's go into more detail on what the Split-T actually was. To do that, we're going to revisit a series I put together back in 2015.
I. Football never changes
Since the author has in a manner of speaking grown up with the "Split T," he has in the process acquired certain convictions about its offensive value. He has four basic reasons for preferring this system.
1. It requires only average personnel.
2. It averages more yards per play.
3. It springs the backs into the open field more often.
4. It puts greater pressure on standard defenses.
II. The play sequence and other technical details
III. The staples
Again, football never changes. You needed both efficiency and explosiveness in 1950, just as you do in 2015. The methods for accomplishing that have changed only as defenses and the physical capabilities of the players themselves have changed.
IV. The constraints
The success of the Fullback Counter, like that of the sneak, is not measured in terms of its offensive average. This is true in spite of the fact that the play has averaged a commendable 4.7 yards at Missouri over a three-year period.
The fullback counter is utilized as a check play to "freeze" the defense and give the basic series more latitude. The play often results in long yardage, especially when a fast, hefty fullback shakes himself loose in the secondary.
In contrast to the sneak, the counter play is not recommended for short yardage or for goal-line thrusts because of the delay element involved. Delayed plays are usually duck soup for the fast, converging line usually encountered near the goal. Faster-hitting straight plays or wide sweeps are the answer to tight defenses.
V. In which Don Faurot stretches the Split T into a spread
The spread formation sets the right end approximately ten yards away from his tackle. The left half flanks to a position about 10 yards outside and one yard behind the offensive left end. The fullback and the right halfback are in normal position. This widens the defensive halfbacks more than does the single flanker and also uncovers more pass territory. [...]
Since two men on each side are eligible as receivers, these formations are excellent ones from which to pass. Only a limited number of plays can be run with standard blocking. ... If a defense spreads its linebackers to meet the spread formation it will be weaker down the middle.
VI. Let's talk blocking and adjustments
When college football began, it was a "three yards and a cloud of dust" sport. Even when the rules adjustments of the early 20th century opened the game up a bit to decrease injuries, it was still power über alles. The option allowed a little bit of misdirection into the equation, and over time, blocking became less about pushing your guy over and more about keeping your guy out of the runner's way. Since the Split T was originally conceived as a way to make up for disadvantages in size and athleticism, it makes sense that this would be the goal of the blocking, too.
The timing of the war, Faurot's generosity in sharing his ideas, and his insistence on limited recruiting all conspired to hold him back to some degree at Mizzou. But Faurot's progressive thought, along with his creativity in combining a lot of influences into something truly unique, changed college football. And some of the principles he formed around this time still actively influence the modern game.


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Dear sisters:
"… what we have seen and heard we proclaim now to you, so that you too may have fellowship with us; for our fellowship is with the Father and with his Son, Jesus Christ. We are writing this so that our joy may be complete" (1 Jn 1, 3-4).
This is precisely what we have experienced and expressed unanimously at the conclusion of our PC. We are all very happy with the fraternal atmosphere of respect, listening, discernment and commitment to the task entrusted to us. We have felt that the Lord continues to encourage us to live this charism that He gave us through Candida María de Jesús.
Today we elected the members of the editorial team, Srs. Teresa Ramírez and María Fuencisla Sanz.
After sharing our evaluation permeated by thanksgiving, our Provincial Superior, with some words that gathered the grateful feeling of all, declared the Provincial Congregation closed.
After a time of rest, we closed the day with the Eucharist, thanking God for the grace received in these days; and we continue praying to live our life in discernment, always open to listening to the Spirit. We are committed to our prayer for the 18th General Congregation.
We want to let you know that we received the visit of the management team of Colegio Reina de la Paz, who, after the initial greeting, delighted us with some songs and gave us a beautiful institutional agenda since they are celebrating the 50th anniversary of the School.
We want to give special thanks to the Community of Reina de la Paz and the Colombia-Venezuela Region, as well as to the ladies and nurses who, with such dedication, take care of the sisters. We have witnessed the silent, joyful, gratuitous and dedicated service of all of them, an expression of the Gospel of Jesus.
You can watch photos of the Provincial Congregation in this link
Information Team


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As an area in the southern part of Ontario, Kitchener acts as go to the place of the Regional Town of Waterloo. Its city consists of the surrounding cities of Waterloo as well as Cambridge, and currently has regarding 220,000 citizens within its own urban area frontiers. While its border metropolitan area of Waterloo is enhanced by 2 universities, the urban area of Kitchener is additional of a blur collar community, and also is house to four service playgrounds: the Bridgeport Business Park, Huron Business Playground, Lancaster Corporate Center, and the Grand Stream West Organization Playground. Hence, Kitchener is actually home to seat manufacturers as well as furniture parts producers. While this is true, some of the old commercial companies located in Kitchener have fallen on unfortunate times, as well as have shut or even quit creation in Kitchener in favor of licensing or even supply arrangement somewhere else. Because of this, it is certainly not completely an unpleasant surprise that some individuals staying in Kitchener falls on hard times as well, and find themselves wanting a bit of financial backing every now and then.
Payday car loan Kitchener is actually one service that homeowners of Kitchener can most likely to in times of financial constraints. Among one of the most pleasing offerings of payday advance loan Kitchener is actually that unlike significant lending providers that no question is actually a lot more well-known withthe basic populace, it is actually very beneficial, fast, and also reliable. As a matter of fact, hold-ups are actually very seldom complied withwhen making an application for a car loan. Within an hour of passing a request, your finance is going to be actually authorized without any issue of whether you possess really good or even quick loans for bad credit. You also won' t undergo any humiliating history inspection, and also you can easily even carry out the use in the very benefit of your very own residence.
Payday Lending in Kitchener Are Readily Available 24/7
If you stay in Kitchener and you need prompt cashmoney, then payday advance loan Kitchener is actually undoubtedly the answer to your financial trouble. The absolute most you will definitely need to perform to obtain cashadvance Kitchener is to fill in an online form that is actually quickly accessible online. You succeeded' t even need to have to leave behind the conveniences of your residence! They also have very marginal needs: be at the very least 19 years of age, a Canadian person or even resident of the nation, have a stable job for at the very least pair of months, an active bank account, and also be readily accessible for calls at a particular phone number. What' s a lot more is actually that your finance is going to be permitted within an hour of slip and also payday advance Kitchener are going to online transfer the money you need to have to your bank account, and also be ready for your usage within the next 24-hour.
Payday funding Kitchener uses the absolute most beneficial as well as fastest settings of amount of money providing for individuals in Kitchener who find themselves captured in an unwarrantable monetary condition. They possess customer support agents accessible online 24/7 to address your questions as well as manage to talk about withyou every little thing to perform withyour lending. This solution is actually additionally able to use affordable rate of interest as well as you might also choose to choose to have your financial obligation repayment opportunity quick loans for bad credit over a couple of paychecks if you thus decide on.


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11 September, 2019
Gemmy Halloween Airblown Inflatable 10ft Cemetery Scene AS IS (READ DESCRIPTION)
Posted in : gemmy on by : admin Tags: 10ft, airblown, cemetery, description, gemmy, halloween, inflatable, read, scene
Gemmy Halloween Airblown Inflatable 10ft Cemetery Scene AS IS (READ DESCRIPTION). Hello there, you are looking at the Gemmy Halloween Airblown Inflatable 10ft Cemetery Scene. This Inflatable is 10ft long and about a little over 9ft tall, I dont exactly know how wide it is, but its pretty wide. The condition of the fabric looks very good, it doesnt really have any fading other than the ghosts being very slightly yellowed in some spots. The inflatable itself doesnt really inflate inflate the greatest as some parts dont really inflate that good (mainly the opposite side of where the blower is) , but its still useable it just needs to be tethered good because the archway has trouble standing up on its own (see pic 6 for an example). (I would recommend getting a second motor for it because inflates very good and basically perfectly with two blowers) see the last pic aka pic 12 for details. (Other motor not included sorry). From what I understand, this an older gemmy lightshow Inflatable, it has flashing lights and a soundbox. Unfortunately the sound DOES NOT work and I have no idea what is wrong with the soundbox. Some flashing lights could be replaced as not all of them work. This inflatable has some small holes (see pics 8 and 9 for details). This inflatable DOES have the original box (see pics 10 and 11 for details). The spider spins the cocoon man and that works. Overall this inflatable is quite a unique Halloween decoration, while it may not be in 10 out of 10 condition, it still has some life left in it and it would make a great addition to your yard this Halloween Season! The item "Gemmy Halloween Airblown Inflatable 10ft Cemetery Scene AS IS (READ DESCRIPTION)" is in sale since Saturday, August 24, 2019. This item is in the category "Collectibles\Holiday & Seasonal\Halloween\Current (1991-Now)\Yard Décor". The seller is "jeanne333music" and is located in Bangor, Pennsylvania. This item can be shipped to United States, Canada, United Kingdom, Denmark, Romania, Slovakia, Bulgaria, Czech republic, Finland, Hungary, Latvia, Lithuania, Malta, Estonia, Australia, Greece, Portugal, Cyprus, Slovenia, Japan, China, Sweden, South Korea, Indonesia, Taiwan, South africa, Thailand, Belgium, France, Hong Kong, Ireland, Netherlands, Poland, Spain, Italy, Germany, Austria, Bahamas, Israel, Mexico, New Zealand, Philippines, Singapore, Switzerland, Norway, Saudi arabia, Ukraine, United arab emirates, Qatar, Kuwait, Bahrain, Croatia, Malaysia, Chile, Colombia, Costa rica, Panama, Trinidad and tobago, Guatemala, Honduras, Jamaica, Cayman islands, Uruguay, Dominican republic, El salvador.
- Brand: Gemmy


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Criminal Record Checks mandatory for all FMHA volunteers
All volunteers must have a completed Criminal Record Check (CRC) / Vulnerable Persons Check (VPC) on file with FMHA before stepping on the ice or bench. They can take 4 to 6 weeks to process so start now for the Sept 01 deadline.
CRC's are not automatically shared between organizations, so if you did one for another organization you still need to do one for FMHA. They are valid for up to 3 hockey seasons and expire on July 31st.
Click on the following link to the BC Justice Institute to complete your CRC electronically. Please use this method rather than going to a local police station as it it much more time efficient and better for record keeping. Most police stations are no longer doing CRC's for volunteer positions. If the system is unable to identify you electronically it will prompt you to follow the instructions and complete and print the application form. If this happens you will then need to contact Lisa Samycia, FMH President at firstname.lastname@example.com to have your photo id verified and the application submitted.
If you have a current valid CRC done with the Justice Institute for another organization, you may choose the "share my results" option during the initial step of the application process, rather than starting from scratch.
Online link: https://justice.gov.bc.ca/eCRC/
FMHA Access Code: TLRUFEGRSS
Once your CRC application is submitted, send your Session ID / confirmation # to firstname.lastname@example.com along with your full name and Date of Birth so that we know your application is in process. You cannot volunteer for FMHA without proof of this.
If you have any questions or are unsure if your past CRC is still valid, please contact Lisa Samycia at firstname.lastname@example.com or 250-423-1054.


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The Office of Prescription Drug Promotion (OPDP), the Food and Drug Administration's pharmaceutical advertising watchdog unit, has issued a warning to a company for allegedly promoting its product Injectafer for uses for which it had not received FDA approval.
The warning, an Untitled Letter, is less serious than the agency's more formal Warning Letters. The untitled letter does not threaten immediately regulatory enforcement action if a company fails to comply, according to Regulatory Affairs Professional Society (RAPS).
The letter, the first to be issued in 2015, was send Luitpold Pharmaceuticals in New Jersey, concerning Injectafer, an injectable treatment for adults with anemia who have not been successfully treated with oral iron or who have non-dialysis dependent chronic kidney disease.
OPDP explained that a video advertisement submitted by the company to regulators contains claims that could be interpreted to mean that Injectafer "is intended for a new use for which it lacks approval, and for which its labeling does not provide adequate directions." The FDA said such claims would cause the drug to be misbranded under federal law, RAPS reports. The FDA contends that the ad talks about iron deficiency anemia (IDA) in general terms, and without necessary caveats about the limitations of Injectafer's intended use. According to the FDA, portions of the ad are problematic from a regulatory perspective because they represent the drug as being intended to treat "all patients, including children, with IDA." The FDA noted that the ad presented Injectafer's two approved indications as add-on conditions. The FDA felt Luitpold's ad had minimized potential risks associated with the product.
The risks are presented in text format at the bottom of the screen for just 30 seconds in the seven-minute ad segment. In addition, the FDA said the text was difficult to read because it was not presented on a contrasting background, according to RAPS. OPDP requested that the company immediately withdraw the advertisement.


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In John David Mann's and my book, The Go-Giver, the foundational principle is the providing of value. It isn't the only thing, but it's certainly the block upon which all else is built.
We're often asked how exactly one — either as a business owner or employee in a company — can build value for their customer and, naturally, it depends upon the type of business.
When really considering it, there are thousands of ways. In Go-Givers Sell More, however, we break it down into five distinct areas: Excellence, Consistency, Attention, Empathy and Appreciation.
In a recent two-part series over at TheGoGiver.com blog entitled, "Ernesto Would Be Proud" I share an example of my favorite local restaurant and how they incorporate the above five.
While at the Palm Beach International Airport recently I experienced an example of how another organization can both apply and not apply the concept of Empathy.
We frequent travelers experience both good and bad examples of value — including empathy — from the various TSA employees conducting the screening process. Usually, however, it's not such a dramatic contrast in such a short period of time.
On my way through I was a bit nervous about the damage that might be caused by the screening machine to the many DVDs I had with me. These were videos of past presentations that I was taking with me to North Carolina to edit into a new video; some of my life's work! Would the machine damage it? Logically, I didn't think it would but, being human and searching for reassurance from the woman conducting the operation, I thought I'd ask.
Me: Excuse me, is there any chance these DVDs could get damaged when going through?
Her: No.
Me: I'm sorry, you're totally certain?
Her: Yes.
Me: Any chance at all I might be able to simply hand them to you without putting them through?
Her: No.
Me (Now realizing I'm sounding a little stupid): So, I have to put them through.
Her: Yes.
Since it's difficult to read emotion into written dialogue, picture me asking extremely politely but with the angst of a person concerned about some of his life's work being ruined and her being — in no way mean but — totally expressionless, emotionless, uncaring and unempathetic as to what I was feeling.
Just then, another TSA employee approached me and said, "Sir, I heard what you were asking. Please be assured, this is an area I'm very familiar with. There will be absolutely no damage to your DVDs. While there are some very specific types of camera films this could possibly happen to, the chances are zero with what you have right there. It's going to be fine."
Wow! Wowee Wow! What a difference! What a huge difference!
In the next article, we'll look at the "key to communicating empathy." Oh, if you are wondering if there was a line behind me I was holding up, no; I was the only one in line. Wouldn't want you to think I was self-centeredly holding up others. Of course, I was indeed being self-centered but was not holding up others.) 🙂
So, how do you apply empathy in your business in order to add value to the relationship? And, do you have any positive examples of others who have done this? Please feel free to share.
Enjoy this post? Receive an update when our next post is published by entering your best email address below and clicking Get Updates.


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Home About the author Gentleman crusader List of articles Books Jamila Verghese
Gandhi has alas been ritualised and rendered largely irrelevant by pious Gandhians who have frozen him in time, reduced him to an ism and have failed to relate his essential truths to contemporary issues
Gandhi has been ritualised and frozen. Yet he was a dynamic force. Change is good. Parties need to nurture younger cadres and hear new voices.
By B G Verghese
Sahara Times, 2006
Years ago there was a famous Shankar cartoon depicting Gandhi striding along briskly, staff in hand, behind a ragged crowd. The caption read: "There go my people. And I must hurry to follow them. For I am their leader"! One must be prepared to follow to be able to lead. This does not mean following the fool multitude. What it implies is the need to understand the problems of the people, articulate their aspirations but yet guide and mentor them to work for a larger purpose. It was to understand and empathise with his people that the Mahatma adopted the dhoti and chadar, which was all they could afford and, for the most part, needed to wear.
One was reminded of Gandhi on January 30, Martyrs Day, when ritual homage was paid to his memory. Few remember the values he stood for; fewer seem to care. One also could not but recall his concept of leadership a few days prior to that event with sundry Congressmen, not all of them necessarily young, clamouring for Rahul Gandhi's elevation to high party office during the AICC's Hyderabad session.
Gandhi has alas been ritualised and rendered largely irrelevant by pious Gandhians who have frozen him in time, reduced him to an ism and have failed to relate his essential truths to contemporary issues and distil an agenda for action. Yet India and the world need Gandhi in our midst today. People abroad have sought to reach out to him more creatively than most of us in India.
Gandhi was dynamic and moved with the times, always ready to reinterpret and adapt, even disown, yesterday's verities in facing current realities. The khadi and village industries movement was developed by him as a strategy to put idle hands to work and achieve mass production through production by the masses. The charkha was only a symbol, not an end product in itself as it has become, at odds with technology and modern management and marketing techniques with backward and forward linkages. Few realise that the KVIC controls the largest retail chain in India which, imaginatively used, could be made a vehicle for rural transformation.
The discovery by corporate houses of a huge market in rural India and the e-chaupal illustrates something of the potential waiting to be tapped. Kamla Chaudhury, a management guru touched by Gandhi, sadly passed away recently. She spent her last years thinking of how to spark a latrine movement to improve sanitation, bestow dignity in the underprivileged, deliver gender justice and promote better health. Barring Bindeshwar Pathak of the Saulabh Shauchalya and bhangi mukti movement and some older Gandhians in the Sabarmati Ashram, where are the Gandhians in all of this ?
Surely it is time for the country to rethink what Gandhi should mean to us over and beyond the stereotyped observance of October 2 and January 30. And have Congresspersons become so forgetful of their heritage as to be unable to rise above thinking about the loaves and fishes of office, often for personal pelf and power, rather than for service to the nation?
Fortunately Rahul Gandhi conducted himself admirably in stating politely, yet firmly, that he is no neta and has much to learn before aspiring to a leadership thrust upon him by sycophants. Indeed, he has to mature and earn his place in the Party.
The notion that young MPs, MLAs or Party workers have no role to play unless they are in "office" must be rubbished. Legislators must learn to know and serve their constituencies and to specialise in matters on which they can bring inquiring minds to bear. Asking Questions and participating in debates and in the proceedings of various parliamentary committees are important responsibilities. It is then for them to lobby for policies and programmes within the Party, in Parliament and within the Government and to build public opinion to further these causes. This does not require ministerial or party positions or all manner of official paraphernalia.
All Parties must also nurture younger cadres by enabling them to make their views known in internal fora or by writing papers which are then given due consideration. There is no reason why youthful members should not be given ministerial office if their talents so warrant. However, it is a pity that Deputy Ministers have virtually disappeared and all the greater regret that the practice of appointing parliamentary secretaries to assist senior minister has been altogether abandoned. These positions carried no perks but offered a valuable training ground to garner political experience and opportunity to prove one's mettle.
Dr Manmohan Singh could do well to consider reintroducing this system but without having the need to consult with coalition partners in terms of numbers, the persons selected and the ministries to which they might be attached as in the case of Ministers – a pernicious spoils system that has weakened the position of the prime minister in recent coalitional politics. Nor should it be necessary to pump up MPs through such devices as the MP Local Area Development Fund, which randomly fragments budget allocations a the district level in accordance with individual whim and fancy and has created a market for patronage in place of service and the true cultivation of constituencies. The recent exposure of misuse of these funds only underlines the point.
It would also be desirable for political parties and citizen groups to try and build up constituency committees that could monitor party activity at the grassroots level and serve as a sieve at the time of selection of electoral candidates. It might not be possible to replicate the American primaries; but some method of screening would be useful in keeping out candidates with dubious records seeking a parliamentary launch pad.
See also AsianConversations.com
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The European Union (EU) is to co-finance the roll out of European Rail Traffic Management System (ERTMS) along a section of high-speed railway in Italy.
Around €4.5 million will be provided from the TEN-T Programme to help fund two projects designed to ensure the full interoperability of a rail corridor connecting Sweden, Germany, Austria and Italy.
The first, which the EU is contributing €1.4 million to, will involve upgrading the current in-cab ERTMS software on 12 Frecciarossa 1000 high-speed trains to conform with the latest version of the system.
The second will see the installation of ETCS trackside equipment along a section of the line between Torino and Napoli.


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The Permian is usually broken into Lower and Upper subdivisions. The faunal stages from youngest to oldest are:
Sea levels in the Permian were low and near shore environments were limited by the collection of almost all major landmasses[?] into a single continent -- Pangea. One continent - even a large continent - has less shoreline than six to eight smaller ones. The Permian ended with the most extensive extinction event recorded in paleontology. 90% to 95% of marine species became extinct. There is very modest evidence that the extinction was caused by weather changes[?] due to impact by a large meteorite. Land life in the Permian included diverse plants, large amphibians and large reptiles including the ancestors of the dinosaurs. The first modern trees - conifers - appeared in the Permian. Permian marine deposits are rich in mollusks, echinoderms, and brachiopods. The last trilobites died out before the end of the Permian.
During the Permian, all the Earth's major land masses except portions of East Asia were collected into a single landmass known as Pangea. Pangea straddled the equator. Deserts seem to have been widespread in Pangea.
The Permian Basin in the U.S. state of Texas is so named because it has one of the thickest deposits of Permian rocks in the world.
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Woodworking mistakes are actually good for you. A mistake gives you the opportunity to learn. This rarely happens when everything goes well. Yes, you do learn with everything you do, but a mistake gives you an opportunity if you look at it the right way.
If You Like My Posts, You'll Love My Books
Why Mistakes are Good in Woodworking
I have done the vast majority of my learning through making mistakes. Not having a teacher, and relying on woodworking books and looking things up online has afforded me several woodworking mistakes.
The way you handle a woodworking mistake is where you can decide to make it something beneficial. After all, if a piece was ruined then you need to find a silver lining somewhere.
Beginning woodworkers tend to see mistakes as failures. They think that they cannot be a good woodworker if they make mistakes. This cannot be farther from the truth. Every good woodworker has made many mistakes in their careers. Even current woodworkers still make mistakes after years of practice. The difference is that they are better at pivoting than they were in the beginning. Hiding the errors or pivoting the project slightly are something that you get better at with time.
The best form of learning is by doing. It is through the act of practice that most woodworking mistakes occur. These afford you more of an opportunity to learn about the craft than at any other time. They also teach you little changes you can make to your designs that turn your mistakes into design opportunities.
There are some strategies that can help you cope with the inevitable woodworking blunders that will come your way. First, beginners should expect to make many mistakes.
Come See What I'm Making on Etsy!
Divorce yourself from the idea that you will hit a home run on the first try. People who can do that are rare, and people who think they can do it every time are delusional. Expect some failures in the beginning, and they will not be as big of a hit when they come.
Second, make sure that you are learning something from your mistake. The first time you do something wrong it's called a mistake, the second time you do the same thing wrong it's called a choice. When you know the wrong way of doing something through trial and error, then you also know the right way too.
Once you know that brad nails don't work like real nails, you will never forget the glue again. If you do, then you are making a decision, not a mistake. We all make mistakes, the key is not to make the same mistake a second time.
Study your mistakes. Make sure that you completely understand why things did not go as you had intended. Once you have a thorough understanding, you then have the ability to try again without making the same error.
Woodworking mistakes will teach you, and it can take years and years of making mistakes until you have made enough to call yourself a good woodworker. Eventually, you will have done enough things wrong that you know the way to do them right by default.
That sounds bad, I know. However, if you are making a project several times (like a guitar maker, or furniture maker) you will have many different disciplines to learn. Each of those has their own woodworking mistakes that can happen along the way. As you make small mistakes here and there, it strengthens your ability and skill. You will come to a deeper understanding of what you are doing, and will be able to create better and better projects.
As for the random pictures…it's confession time. The Thor Hammer has a couple bricks inside the head. I thought it would be a good idea to make it heavy like the movie. Not only is it a pain to show people, but it's also a pain to lug around. I gave it to my brother in-law for a display piece, and that's the best thing for it.
The pipe looks beautiful, but you can't smoke it. The laminated wood has glue in the tobacco chamber, which would not be good to smoke. Lastly, the inlay on the wine ceremony box was under filled, which means I had to fill it a second time to get it level. I'm not proud of my mistakes, but I have definitely learned from them.
Come See What I'm Making on Etsy!
The moral of the story is not to worry. Mistakes are going to happen, and that's ok. Spend your time learning as much as you can. Destroy some wood, and make sure that you come out with more knowledge on every project. It's only wood, you can buy more.
If you have any questions on Woodworking Mistakes and Why They Are Good For You, please leave a comment and I will be glad to answer them. Also, please share my work with your friends on Pinterest. Happy building!
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pvt.fm Outstanding waterfront home, wide water, ocean access, newer dock, Dual lift for jet ski and boat. Can be sold fully furnished including working Seadoo 22FT. and Seadoo GTX Jet ski on site. Easy to show, very motivated seller. Dream vacation home or residence in desirable Marco Island Paradise. Close to restaurants and shopping area, 5 minute drive to beaches. Don't miss showing and selling this home. Bonus available. | From:MiamiAssociation Views:0 0ratings | |
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Excellent Education in a Faith-Filled Community
In Grade 2, we focus on the two Sacraments, First Reconciliation and First Eucharist. Both are parish sacraments and St. Vincent Martyr Christian Formation program handles some of the preparation. However, the focus of our Religion program in Grade 2 is on Reconciliation and Eucharist and the students are very well prepared to receive them.


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Adipose tissue is a pivotal organ determining longevity, due largely to its role in maintaining whole-body energy homeostasis and insulin sensitivity. SIRT1 is a NAD-dependent protein deacetylase possessing antiaging activities in a wide range of organisms. The current study demonstrates that mice with adipose tissue–selective overexpression of hSIRT1(H363Y), a dominant-negative mutant that disrupts endogenous SIRT1 activity, show accelerated development of metabolic aging. These mice, referred to as Adipo-H363Y, exhibit hyperglycemia, dyslipidemia, ectopic lipid deposition, insulin resistance, and glucose intolerance at a much younger age than their wild-type littermates. The metabolic defects of Adipo-H363Y are associated with abnormal epigenetic modifications and chromatin remodeling in their adipose tissues, as a result of excess accumulation of biotin, which inhibits endogenous SIRT1 activity, leading to increased inflammation, cellularity, and collagen deposition. The enzyme acetyl-CoA carboxylase 2 plays an important role in biotin accumulation within adipose tissues of Adipo-H363Y. Calorie restriction prevents biotin accumulation, abolishes abnormal histone biotinylation, and completely restores the metabolic and adipose functions of Adipo-H363Y. The effects are mimicked by short-term restriction of biotin intake, an approach potentially translatable to humans for maintaining the epigenetic and chromatin remodeling capacity of adipose tissues and preventing aging-associated metabolic disorders.
Advancing age is associated with loss of energy balance and deterioration of metabolic functions. In older people, postprandial hyperglycemia is common (1). The prevalence of glucose intolerance and insulin resistance increases progressively and significantly with age (2). The redistribution of fat from subcutaneous depots to visceral compartment and from adipose tissues to ectopic sites plays a critical role in the development of aging-related metabolic abnormalities, in particular type 2 diabetes. Older people exhibit elevated central fat deposition but decreased skeletal muscle mass (referred to as sarcopenic obesity) (3). Preadipocytes isolated from aged animals and humans have a reduced potential to differentiate and replicate, whereas aged adipocytes show a decreased capacity for lipid storage (4). The mechanisms underlying age-associated changes in adipose tissue function and fat distribution remain incompletely understood.
SIRT1 is a mammalian ortholog most closely related to SIR2 (silent information regulator 2), a protein identified in Saccharomyces cerevisiae that extends replicative life span (5). Both SIRT1 and SIR2 belong to a conserved family of aging regulators and are NAD-dependent deacetylases that catalyze the removal of acetyl groups from protein substrates (6). In yeast, SIR2 acts as a transcriptional silencer by modulating histone modifications, especially at the mating-type loci and telomeric DNA regions (7). These chromosome domains are analogous to the heterochromatin of multicellular eukaryotes. When a conserved histidine within the catalytic domain of SIR2 was converted to a tyrosine (H364Y), its histone modification activity was lost and the mutant acted in a strong dominant-negative manner (8). Compared with SIR2, SIRT1 elicits a wider range of biological functions by interacting with and regulating diversified protein substrates (5).
Animal studies reveal that SIRT1 is a key energy sensor controlling cellular responses to nutrient availability and in turn protecting against aging-related metabolic diseases (9). SIRT1 is critically involved in the metabolic adaptation to fasting, exercise, and calorie restriction (10), which upregulate the activity of this antiaging protein in adipocytes and adipose tissues (11). Activation of SIRT1 in turn stimulates fat mobilization and adiponectin production in adipose tissues (12–14). Overexpression of SIRT1 in adipose tissues of mice induces a phenotype similar to that with calorie restriction (leaner and metabolically more active, with improved insulin sensitivity and glucose tolerance [14–16]). This evidence suggests that adipose SIRT1 is a candidate drug target for promoting systemic energy homeostasis and healthy aging.
Transgenic mice that selectively overexpress wild-type (WT) human SIRT1 (hSIRT1) or hSIRT1(H363Y) mutant in adipose tissues have been established (14). In the current study, the metabolic functions of these two types of transgenic mice, referred to as Adipo-SIRT1 and Adipo-H363Y, respectively, are compared and related to those of the WT controls. Whereas Adipo-SIRT1 are more insulin sensitive and metabolically healthier than their WT littermates (14), Adipo-H363Y exhibit a phenotype of accelerated metabolic aging. Calorie restriction has been applied to test whether or not this nongenetic intervention is able to restore a normal metabolic function in Adipo-H363Y. The results demonstrate that a large number of aging-induced changes of gene expression in adipose tissues are prevented by calorie restriction, which significantly delays the development of insulin resistance, glucose intolerance, and dyslipidemia in both WT and Adipo-H363Y. Limited biotin intake contributes to the beneficial effects of calorie restriction on enhancing SIRT1 activity in adipose tissues and improving systemic energy homeostasis. At the epigenetic level, by preventing excessive histone biotinylation, calorie restriction facilitates SIRT1-mediated histone deacetylation and transcriptional silencing.
Research Design and Methods
Animal Experiments
All procedures were approved by the Committee on the Use of Live Animals for Teaching and Research of The University of Hong Kong and carried out in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) as well as institutional guidelines for the care and use of laboratory animals. The generation of transgenic mice with selective overexpression of human SIRT1 (Adipo-SIRT1) or hSIRT1(H363Y) (Adipo-H363Y) in adipose tissues was previously described (14). Mice were housed in a room under controlled temperature (23 ± 1°C) and 12-h light-dark cycles, with free access to water and standard mouse chow (4.07 kcal/g composed of 20% protein, 52.9% carbohydrate, 5.6% fat, 4.7% fiber, 6.1% minerals, and vitamin mixture containing 0.3 mg/kg biotin; LabDiet 5053; LabDiet, Purina Mills, Richmond, IN). All animals appeared normal and were fertile, giving rise to healthy litters. Male mice were used for the current study.
The food intake for ad libitum–fed mice was ∼0.14 g/g body weight/day. For calorie restriction, mice received 60% of the normal food supply (∼0.08 g/g body weight/day) from the age of 10–40 weeks. To study the effects of biotin intake in the diet, mice were given either biotin-sufficient (4.11 kcal/g composed of 18% casein, 60% sucrose, 10% vegetable oil, 2% corn oil, 4% salt, and vitamin mixture containing 0.45 mg/kg biotin; 02960407; MP Biomedicals, Santa Ana, CA) or biotin-deficient (4.11 kcal/g composed of 18% casein, 60% sucrose, 10% vegetable oil, 2% corn oil, 4% salt, and vitamin mixture without biotin; 02901030; MP Biomedicals) diets. Mice were randomly assigned to each experimental group (n = 8–10) for diet treatment starting at the age of 10 weeks. Metabolic parameters were monitored on a weekly basis.
For in vivo RNA interference (RNAi) treatment, three sets of chemically modified silencing RNAs (siRNAs) for acetyl-CoA carboxylase (ACC)2 were synthesized by RiboBio (Guangdong, China), including sense 5′-CCUACGAGAUGUUCCGUAAdTdT-3′/antisense 3′-dTdTGGAUGCUCUACAAGGCAUU-5′, sense 5′-GCAUCAAGUAUGCUCUCAAdTdT-3′/antisense 3′-dTdTCGUAGUUCAUACGAGAGUU-5′, and sense 5′-CCACCUAUGUGUACGACUUdTdT-3′/antisense 3′-dTdTGGUGGAUACACAUGCUGAA-5′. Mice were treated with the mixture of ACC2 siRNAs (5 nmol/mouse/day) or the same amount of scrambled RNAi by intraperitoneal injection at six different sites for three consecutive days (17). The epididymal adipose tissues were harvested 12 h after the last injection for subsequent analysis.
Metabolic Evaluations
Body weight, blood glucose, and fat mass composition were measured between 1000 and 1200 h for mice that were either starved overnight or fed ad libitum. Blood glucose was monitored by tail nicking using an Accu-Check Advantage II Glucometer (Roche Diagnostics, Mannheim, Germany). The body composition was assessed in conscious and unanesthetized mice using a Bruker minispec Body Composition Analyzer (Bruker Optics, Inc., Woodlands, TX). The intraperitoneal glucose tolerance test (ipGTT) and insulin tolerance test (ITT) were performed as previously described (14,18). Circulating and tissue contents of lipids, including triglycerides, total cholesterols, and free fatty acids, were analyzed using LiquiColor Triglycerides and Stanbio Cholesterol (Stanbio Laboratory, Boerne, TX) and the Half-Micro Test Kit (Roche Diagnostics), respectively. The fasting serum insulin concentration was quantified using the commercial ELISA kits from Mercodia AB (Uppsala, Sweden). Serum adiponectin and lipocalin-2 concentrations were determined with the in-house ELISA kit as previously described (19,20). Metabolic rate (VO2, VCO2, and respiratory exchange ratio [RER]) was measured by indirect calorimetry using a six-chamber open-circuit Oxymax system component of the Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH) as previously described (14). All mice were acclimatized to the cage for 48 h before recording the parameters during a 2-day feeding and a 24-h fasting period.
Microarray Analysis
Total RNA was extracted from epididymal adipose tissues of WT, Adipo-SIRT1, and Adipo-H363Y mice aged 8 and 40 weeks using the RNeasy Mini Kit (Qiagen, Valencia, CA). Samples were derived from at least three mice in each group. The quality of RNA was assessed by a spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington, DE) and the RNA 6000 Pico LabChip with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The A260-to-A280 ratios of all RNA samples were between 1.9 and 2.1. The RNA integrity numbers were >9. After reverse transcription, the labeled cDNA samples were hybridized to the Mouse Genome 430 2.0 expression arrays using the GeneChip Fluidics Station 450 (Affymetrix Inc., Santa Clara, CA). The intensity of the probed signals was scanned and captured by GeneChip Scanner 3000 (Affymetrix Inc.). Quality of the raw intensity data was assessed using diagnostic plots by GeneSpring GX 12.6 (Agilent Technologies) and Affymetrix Expression Console (Affymetrix Inc.). Raw intensity values of gene expression were normalized and background corrected using the Robust Multiarray Average algorithm. Genes with expression values lower than the 20th percentile of all intensity values were excluded from further analyses. The expression profiles of samples derived from WT, Adipo-SIRT1, and Adipo-H363Y were clustered and validated by principal component analysis. Data comparisons were performed between 1) 8-week-old WT, Adipo-SIRT1, and Adipo-H363Y; 2) 40-week-old WT, Adipo-SIRT1, and Adipo-H363Y fed ad libitum; 3) 8- and 40-week-old ad libitum–fed mice of the same genotypes; and 4) 8- and 40-week-old mice of the same genotypes under calorie restriction. Differentially expressed genes were defined as more than a twofold change using a generalized linear model. In total, a list of 3,915 genes that were changed in at least one set of the above comparisons is included in Supplementary Table 1 for further analysis. Venn diagrams were used for presenting the numbers of differentially or commonly changed genes. The MIAME-compliant microarray data have been submitted to and are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress, accession number E-MTAB-2777).
Quantitative Reverse Transcription PCR
Quantitation of target genes was performed using SYBR Green PCR Master Mix (Qiagen) and an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). The primer sequences are listed in Supplementary Table 2. Data were calculated and are presented as relative expression of transcripts normalized to β-actin.
Chromatin Immunoprecipitation–PCR
The primers for chromatin immunoprecipitation–PCR (ChIP-PCR) are listed in Supplementary Table 2. The histone acetylation and/or biotinylation at the promoter regions of different genes were analyzed and compared by ChIP-PCR as previously described with modifications (21). In brief, epididymal adipose tissue (100 mg) was minced into small pieces in cold PBS (137 mmol/L NaCl, 4.3 mmol/L Na2HPO4, 2.7 mmol/L KCl, 1.4 mmol/L KH2PO4, pH 7.4) with protease inhibitor cocktails. Formaldehyde was added to a final concentration of 1%, and the mixture was incubated at room temperature for 15 min with moderate shaking. The cross-linking was stopped by adding glycine to a final concentration of 0.125 mol/L. The nuclei were isolated from the tissue lysates and resuspended in lysis buffer (1% SDS, 50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L EDTA, containing protease inhibitors) for sonication to obtain chromatin fragments of 500–1,000 bp. After preclearing with protein G–Sepharose beads (Pierce, Rockford, IL) (blocked with 0.2 mg/mL salmon sperm DNA and 0.5 mg/mL BSA), 50 μg chromatin was incubated with 2 μg antibody overnight at 4°C. Immune complexes were collected on protein G–Sepharose beads (blocked as above), washed, and eluted into 100 μL ChIP elution buffer (1% SDS and 0.1 mol/L NaHCO3). Half of the elutes were reverse cross-linked and the other half diluted 1:10 with lysis buffer to perform the second ChIP with streptavidin-conjugated beads (Pierce) as previously described (22). After elution with ChIP elution buffer, the cross-link was reversed by adding 8 μL of 5 mol/L NaCl and incubating for 4 h at 65°C. The DNA was purified by phenol-chloroform extraction. PCR amplifications were performed using 10% of the DNA samples and the resulting PCR products separated by agarose gel for image quantification by densitometry (MultiAnalyst Software; Bio-Rad). Quantitation was also performed by quantitative reverse transcription PCR analysis. The results were normalized against those from either the input DNA samples or the first ChIP fractions and presented as fold changes.
SIRT1 Activity Measurements
SIRT1 activity was monitored using a Fluorometric Drug Discovery kit (Enzo Life Sciences International, Inc., Plymouth Meeting, PA). Fluorescence was measured with a fluorometric reader (CytofluorII 400 series; Applied Biosystems), whereby excitation was set at 360 nm and emission detection at 460 nm. The in vitro protein deacetylation assay was analyzed by high-performance liquid chromatography as previously described (14). The histone H3 peptides, including ARTKQTARKSTGGK14APRK18QLC with or without acetylation at K14 and/or biotinylation at K18, were used as substrates. In brief, recombinant SIRT1 (3.6 μmol/L) was incubated with peptide substrate (500 μmol/L) and NAD+ (500 μmol/L) in 50 mmol/L Tris buffer (pH 7.5) at 37°C for 10 min, in the presence and absence of 500 μmol/L biotin. Trifluoroacetic acid was added to 0.1% before fractionation by reverse-phased high-performance liquid chromatography on a Symmetry C18 column (5 μm, 3.9 × 150 mm; Waters, Milford, MA), using a 40-min linear gradient (0–40% buffer B containing acetonitrile with 0.5% trifluoroacetic acid) at a flow rate of 1 mL/min. Chromatographic peaks were detected by an ultraviolet detector at 214 nm. The sizes of the peaks obtained by calculating the areas under the peaks were used for comparison.
Quantification of Tissue Biotin Contents
The amount of total biotin (bound and unbound) was determined as previously described (23) with modifications. In brief, 30 mg of tissue or cell lysates was hydrolyzed in sulfuric acid at 100°C for 1 h. After neutralization, 3.5 mg of activated charcoal was added to bind the liberated biotin. The mixture was centrifuged at 12,000 rpm for 3 min, and the bound biotin was released by adding 1 mL of 5% ammonia ethanol solution. The supernatant was filtered and evaporated to dryness under nitrogen. The extract was reconstituted in 100 μL methanol and mixed with 80 μL of 0.1% 9-anthryldiazomethane (ADAM, weight for volume in ethyl acetate) for 1 h at room temperature. The fluorescent biotin-ADAM was detected at an excitation wavelength of 365 nm and emission wavelength of 412 nm using the fluorometric reader. Biotin solutions (1–50 ng/mL) were used to generate a standard curve for calibration and quantification.
Histological Analysis
Paraffin sections (5 μm) were prepared for adipose tissues collected from different groups of mice. Hematoxylin and eosin staining was performed to analyze adipocyte histology under a light microscope (Olympus, Tokyo, Japan). The sizes of adipocytes were measured as previously described (18). Masson trichrome staining was performed using Accustain Trichrome Kits (Sigma-Aldrich, St. Louis, MO) to evaluate the extracellular deposition of fibrillar collagens.
Western Blotting
Antibodies against total ACC, ACC2, histone H3, histone H4, acetylated histone H3 (K9), and acetylated lysine were purchased from Cell Signaling (Beverly, MA). Antibodies against ACC1, SIRT1, and acetylated histone H4 (K5, K8, K12, and K16) were from Millpore (Billerica, MA). Monoclonal antibodies against β-actin and streptavidin–horseradish peroxidase (HRP) conjugate were obtained from Sigma-Aldrich. For Western blotting, proteins derived from cell or tissue lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After overnight blocking, membranes were probed with various primary antibodies followed by secondary antibodies. Immunoreactive antibody-antigen complexes were visualized with the enhanced chemiluminescence reagents from GE Healthcare (Uppsala, Sweden). For histone modification analysis, histones were extracted from the nuclear fractions of epididymal fat pads following a previously reported procedure (21). In brief, the pellets of nuclei were resuspended overnight in 0.4 mol/L H2SO4 at 4°C. The supernatant was collected after centrifugation (16,000g, 10 min at 4°C) and precipitated with trichloroacetic acid at 4°C for 1 h. The acid-soluble histone fraction was subjected to 18% SDS-PAGE and visualized by Coomassie Brilliant Blue staining. The biotinylated protein was detected by Western blotting using streptavidin-HRP conjugate, and the acetylated protein detected with antiacetyl-lysine antibodies.
Data Analysis
The density of protein bands in Western blots was analyzed quantitatively using ImageJ 1.45 software (National Institutes of Health, Bethesda, MD) for calculating the expression ratios against loading controls. Representative Western blotting images are shown. Statistical calculations were performed with the Statistical Package for the Social Sciences version 11.5 software package (SPSS, Inc., Chicago, IL). For multiple comparisons, the differences were analyzed by one-way ANOVA, followed by Dunnett test. Differences in other comparisons were determined by an unpaired two-tailed Student t test. In all cases, statistically significant differences were accepted when P values were <0.05. All results were derived from at least three sets of independent experiments unless otherwise specified. Data are presented as mean ± SEM.
Overexpression of hSIRT1(H363Y) in Adipose Tissue Accelerates the Development of Metabolic Aging
The metabolic parameters, including body weight, blood glucose, serum insulin, and circulating lipid levels, were monitored on a weekly basis. Systemic insulin sensitivity was evaluated by ITTs and ipGTTs. Compared with both WT and Adipo-SIRT1, Adipo-H363Y showed a phenotype of significantly accelerated metabolic aging (Fig. 1). At as early as the age of 20 weeks, abnormal responses to ITT and ipGTT were detected in Adipo-H363Y when compared with age-matched WT (Supplementary Fig. 1). At the age of 30 weeks, the responses of Adipo-H363Y to either ITT or ipGTT were comparable to those of 60-week-old WT (Fig. 1A). By contrast, these responses in 60-week-old Adipo-SIRT1 were not significantly different from mice at the age of 30 weeks. Although the body weight was not significantly different between the three groups of mice at the age of 12 weeks, Adipo-H363Y showed a consistently higher gain of body weights than the other two groups of mice at other age points (Fig. 1B). Fasting glucose levels of Adipo-H363Y were not significantly different from WT (data not shown). However, from the age of 40 weeks onwards, the nonfasting glucose levels were significantly higher in Adipo-H363Y than in the other two groups of mice (Fig. 1C). Circulating insulin levels were rapidly rising with age, being higher in Adipo-H363Y by ∼51, ∼79, and ∼85% than those of WT at the age of 20, 40, and 60 weeks, respectively (Fig. 1D). The insulin concentration in 30-week-old Adipo-H363Y reached a level similar to that in WT at 60 weeks of age. Circulating triglyceride levels augmented progressively in both Adipo-H363Y and WT but remained the lowest in Adipo-SIRT1 (Fig. 1E). Compared with WT, the development of aging-associated hypertriglyceridemia was enhanced significantly by ∼19, ∼21, and ∼31% in Adipo-H363Y at 40, 50, and 60 weeks of age, respectively.
In line with their higher body weight gain, Adipo-H363Y showed significantly increased (by >10%) whole-body adiposity from the age of 20 weeks onwards when compared with WT, whereas changes in opposite directions were observed in Adipo-SIRT1 (decreased by ∼20%) (Fig. 2A). However, the percentage compositions of lean and fluid mass were not significantly different between WT and Adipo-H363Y (Supplementary Fig. 2). On the other hand, the weights of visceral fat pads collected postmortem were not elevated in Adipo-H363Y when compared with those in WT (Fig. 2B), indicating that the excess lipids were deposited in ectopic sites. Indeed, the triglyceride contents in liver and skeletal muscle of Adipo-H363Y were augmented significantly by ∼35 and ∼61%, respectively, when compared with WT (Fig. 2C). In adipose tissues of Adipo-H363Y, the cholesterol and free fatty acid contents were significantly higher (by 1.5- and 1.9-fold, respectively) than in those of WT (Fig. 2C). However, the size of adipocytes in epididymal adipose tissues of Adipo-H363Y was significantly smaller (Supplementary Fig. 3). Histological assessment showed an increased cellularity and trichrome-positive streaks interspersed between adipocytes in adipose tissues of Adipo-H363Y (Supplementary Fig. 4A). The expressions of genes involved in adipogenesis were significantly downregulated, whereas those associated with inflammation were upregulated in adipose tissues of Adipo-H363Y (Supplementary Fig. 4B). Indirect calorimetric analysis revealed that the RER was elevated significantly in Adipo-H363Y during both dark and light periods (Fig. 2D). The increased RER in Adipo-H363Y was attributed mainly to the higher CO2 production (VCO2) but not to differences in O2 consumption (VO2).
Collectively, the results demonstrate that overexpression of hSIRT1(H363Y) selectively in adipose tissues of mice accelerates the development of insulin resistance, glucose intolerance, postprandial hyperglycemia, and dyslipidemia, a cluster of metabolic defects that are closely associated with aging.
Calorie Restriction Restores the Metabolic Functions of WT and Adipo-H363Y
Total SIRT1 protein expression was decreasing with age in adipose tissues of WT but remained higher in those of Adipo-SIRT1 and Adipo-H363Y (Fig. 3A). Compared with that of WT, SIRT1 activity was significantly augmented (by approximately twofold) in adipose tissues of Adipo-SIRT1 but decreased (by ∼60%) in those of Adipo-H363Y (Fig. 3B). Compared with mice at the age of 12 weeks, SIRT1 activities were decreased by ∼35, ∼10, and ∼55% in 40-week-old WT, Adipo-SIRT1, and Adipo-H363Y, respectively. Calorie restriction (60% of ad libitum food intake) for 30 weeks not only prevented the downregulation of SIRT1 activities but also significantly enhanced the latter in adipose tissues of both WT and Adipo-H363Y (Fig. 3C). At the age of 40 weeks, mice under calorie restriction showed similar levels of SIRT1 activity in their adipose tissues, which was elevated by ∼2.1- and ∼4.3-fold compared with the corresponding ad libitum–fed WT and Adipo-H363Y, respectively. Calorie restriction significantly reduced the nonfasting glucose levels in all mice (Fig. 3D) and improved the responses to ITT and ipGTT in both WT and Adipo-H363Y (Fig. 3E). The area under the curve (AUC) values of ipGTT and ITT in calorie-restricted WT and Adipo-H363Y were not significantly different from those of age-matched Adipo-SIRT1 (Fig. 3E, bottom panels). At the age of 40 weeks, the body weight gain and plasma insulin levels in the three groups of mice under calorie restriction were not significantly different (Fig. 3F), and were comparable to those in 20-week-old WT (Fig. 1B and D).
Calorie-restricted WT, Adipo-SIRT1, and Adipo-H363Y exhibited similar diurnal rhythmic patterns of RER, ∼0.82 in dark cycles and ∼0.75 in light cycles (Fig. 4A). The VCO2 of Adipo-H363Y was significantly decreased by calorie restriction in both dark (∼14%) and light (∼23%) cycles, to levels comparable to those in WT and Adipo-SIRT1 (Fig. 4A). The whole-body adiposity was similar in the three types of mice across all age-groups under calorie restriction (Fig. 4B). The percentage weights of postmortem visceral fat pads in calorie-restricted mice at the age of 40 weeks were comparable to those in 20-week-old mice fed ad libitum (Fig. 4B and Fig. 2B). The aging-induced augmentation of circulating triglyceride levels was prevented by calorie restriction in WT and Adipo-H363Y (Fig. 4C). Calorie restriction significantly reduced the lipid contents in adipose tissues of all mice groups (Fig. 4D) and attenuated the ectopic lipid deposition in Adipo-H363Y (Fig. 4E).
Together, these findings suggest that despite the decreased SIRT1 activity in adipose tissues of Adipo-H363Y, calorie restriction can correct both local and systemic metabolic defects caused by adipose overexpression of the mutant hSIRT1(H363Y) and/or aging.
Increased Histone Biotinylation Causes Abnormal Chromatin Remodeling in Adipose Tissues of Adipo-H363Y
Microarray analysis was performed using RNA samples extracted from adipose tissues of mice at the age of either 8 weeks (referred to as "young," when metabolic functions were comparable among the three groups) or 40 weeks (referred to as "old," when significant differences in metabolic functions were obvious). Principal component analysis of the adipose transcriptome revealed that samples derived from young WT, Adipo-SIRT1, and Adipo-H363Y could be separated into three distinct clusters (Supplementary Fig. 5A). The number of differentially expressed (by more than twofold) transcripts was obtained by pairwise comparison between mice of the same age-group or between young and old mice of the same genotype (3,915 genes in total) (Supplementary Table 1). Compared with young WT, 1,390 and 429 genes were differentially expressed in adipose tissues of age-matched Adipo-SIRT1 and Adipo-H363Y, respectively (Supplementary Fig. 5B). Over 90% (1,269) of the altered gene expressions in young Adipo-SIRT1 were due to upregulation (Supplementary Fig. 6A). In adipose tissues of Adipo-H363Y, genes related to cholesterol biosynthesis were significantly upregulated and those of mitochondrial fatty acid oxidation significantly downregulated (Supplementary Fig. 6B). With the progression of aging, different patterns of changes in adipose gene expression were observed in the three types of mice (Fig. 5A). In general, the genome expression and chromatin state in adipose tissues of Adipo-H363Y were more rigid than the other two groups of mice. The average gene expression levels in adipose tissues of young WT, Adipo-SIRT1, and Adipo-H363Y were 0.210, 0.746, and 0.061, respectively. Compared with the corresponding young mice, a significant decrease was observed in the average gene expression level of old Adipo-SIRT1 (−0.081), whereas that of old Adipo-H363Y decreased slightly to 0.041 (Fig. 5A). Compared with those of WT (1,107) and Adipo-SIRT1 (2,425), less (901) numbers of genes were changed by aging (more than twofold) in adipose tissues of old Adipo-H363Y (Supplementary Fig. 7, top panels). In addition to those involved in sterol lipid metabolism, genes encoding extracellular matrix and tight junction proteins were further upregulated in old Adipo-H363Y (Supplementary Fig. 7, bottom panel). A larger number (2,425) of genes were changed in old compared with young Adipo-SIRT1, with ∼70% downregulated, suggesting a more flexible and dynamic chromatin structure in adipose tissues of these mice (Supplementary Figs. 7 and 8). Calorie restriction prevented ∼45 and ∼36% of aging-induced gene changes in adipose tissues of WT and Adipo-H363Y, respectively. Especially, out of the 103 genes upregulated in old Adipo-H363Y adipose tissues, 93 (∼90%) were prevented by calorie restriction (Supplementary Fig. 8, top left panel). Among those that remained unchanged (twofold or less) during aging, 888 and 1,029 were altered by calorie restriction (more than twofold) in WT and Adipo-H363Y, respectively (Supplementary Fig. 7, top right panel). About 47 and 60% of genes changed by calorie restriction (more than twofold) in WT and Adipo-H363Y, respectively, showed similar up- or downregulated patterns of change with those in old Adipo-SIRT1 when compared with young Adipo-SIRT1 (Supplementary Fig. 8, bottom panels).
Histone modifications control the stability and flexibility/rigidity of chromatin structure and its remodeling (24). Thus, histones were purified from adipose tissues of WT, Adipo-SIRT1, and Adipo-H363Y to analyze the modifications at the epigenetic levels. Western blotting showed that even at the young age (8 weeks old), the three types of mice contained different amounts of acetylated and/or biotinylated histones in their adipose tissues. Adipo-H363Y showed the highest level of histone biotinylation, whereas histone acetylation was significantly decreased in Adipo-SIRT1 (Fig. 5B). Coimmunoprecipitation was performed and the results further confirmed that histones, including H3 and H4, were acetylated and biotinylated in adipose tissues of the three groups of mice, with acetylated histones containing significantly higher amounts of biotinylation (Supplementary Fig. 9). Profile plots revealed that calorie restriction decreased the average gene expression levels in both old WT and old Adipo-H363Y (Fig. 5C), which was in line with the reduced histone acetylation levels in both types of calorie-restricted mice (Fig. 5D). In addition, calorie restriction significantly decreased the biotinylation of histones in Adipo-H363Y preparations. By contrast, calorie restriction had no significant effects on histone acetylation and biotinylation in adipose tissues of Adipo-SIRT1, both of which were significantly lower than in those of Adipo-H363Y (Fig. 5D).
To confirm the altered epigenetic modifications in Adipo-H363Y, a number of genes were selected for analysis of the associated histones using ChIP-PCR. The expression of these genes, including CUGBP, GADD, TRDN, and ADIPOQ, was altered by aging and/or overexpression of hSIRT1(H363Y) (E-MTAB-2777 in ArrayExpress). Histone biotinylation was detected at the promoter regions of all four genes, and samples derived from Adipo-H363Y exhibited the highest levels of biotinylated histones (Supplementary Fig. 10, left panels). Consistent with gene expression changes induced by calorie restriction, the amount of biotinylated histones was significantly reduced in samples from calorie-restricted WT, Adipo-SIRT1, and Adipo-H363Y (Supplementary Fig. 10, right panels). The results further demonstrated that the amounts of biotinylated histones were significantly lower in fractions of nonacetylated than in those of acetylated histones (Supplementary Figs. 11 and 12).
Taken together, these results suggest that augmented histone biotinylation contributes to the rigid chromatin structure in adipose tissues of Adipo-H363Y, which prevents the adaptive responses of genomic activation and silencing, thus leading to an accelerated aging phenotype of these mice. The abnormal epigenetic modifications can be largely removed by calorie restriction.
Biotin Deficiency Prevents the Development of Metabolic Dysfunctions in WT and Adipo-H363Y
During aging, biotin is progressively accumulated in adipose tissues of mice and inhibits the deacetylase activity of SIRT1 (14). The biotin content in adipose tissues of Adipo-H363Y was augmented significantly (by approximately twofold) even at the age of 20 weeks when compared with those in WT (Fig. 6A, left panel). Calorie restriction prevented the accumulation of biotin in adipose tissues of both WT and Adipo-H363Y (Fig. 6A, right panel). To further validate the involvement of biotin accumulation in adipose tissues as a cause of systemic metabolic deregulation, WT and Adipo-H363Y were given either a biotin-sufficient or a biotin-deficient diet from 10 to 20 weeks of age. This relatively short period of biotin deficiency did not induce obvious abnormalities but significantly reduced the biotin content and elevated SIRT1 activity in adipose tissues of WT and Adipo-H363Y (Fig. 6B). Compared with those receiving the biotin-sufficient diet, the amount of acetylated and biotinylated histones was decreased significantly in adipose tissues of mice fed the biotin-deficient diet (Fig. 6C). The latter diet also reduced the contents of the vitamin in livers (from 961 ± 113 to 703 ± 83 ng/g in WT and from 1,042 ± 175 to 685 ± 92 ng/g in Adipo-H363Y). By contrast, restriction of total calorie intake did not change the biotin contents in livers of either WT (710 ± 132 ng/g) or Adipo-H363Y (730 ± 111 ng/g), which were comparable to those of mice fed ad libitum (651 ± 98 and 680 ± 88 ng/g for WT and Adipo-H363Y, respectively).
Biotin-deficient mice exhibited significantly improved ipGTT and ITT responses compared with those receiving a biotin-sufficient diet (Fig. 6D). There was no significant difference between WT and Adipo-H363Y. The plasma insulin level was reduced in both WT and Adipo-H363Y fed with biotin-deficient diet (Fig. 6E, left panel). The nonfasting blood glucose was not different between WT and Adipo-H363Y receiving the biotin-sufficient diet, which contained higher amounts of sucrose (60%) and fat (12%) than the standard mouse chow (3.18 and 5.6%, respectively). Nevertheless, after ad libitum feeding with a biotin-deficient diet, the plasma glucose level was reduced in both WT and Adipo-H363Y (Fig. 6E, middle panel). Circulating triglycerides were also decreased by the biotin-deficient diet to similar levels in WT and Adipo-H363Y, when compared with their respective biotin-sufficient groups (Fig. 6E, right panel). Likewise, the triglyceride content in skeletal muscle and liver was significantly reduced to comparable levels in biotin-deficient WT and Adipo-H363Y (Fig. 6F, left panel). Adipo-H363Y under biotin-deficient diet showed an enhanced capacity of triglyceride storage in their adipose tissues. In addition, the free fatty acid contents of adipose tissues were significantly reduced by the biotin-deficient diet in both WT and Adipo-H363Y (Fig. 6F, right panel). Both calorie restriction and the biotin-deficient diet increased SIRT1 protein expression in adipose tissues of WT but did not change that of Adipo-SIRT1 and Adipo-H363Y (Supplementary Fig. 13A). The circulating adiponectin levels were elevated by both calorie restriction and the biotin-deficient diet, whereas the circulating levels of the proinflammatory adipokine lipocalin-2 were reduced by these treatments (Supplementary Fig. 13B and C).
Taken in conjunction, the present results support the concept that biotin accumulation in adipose tissue is causally involved in the development of aging-associated metabolic abnormalities and, thus, that limiting the intake of biotin represents an effective way to enhance adipose SIRT1 function and prevent metabolic aging.
Upregulated Acetyl-CoA Carboxylase 2 Contributes to the Dominant-Negative Effects of hSIRT1(H363Y) on Inhibiting Endogenous SIRT1
SIRT1 regulates the protein stability and acetylation status of acetyl-CoA carboxylase (ACC), a biotin reservoir (14,25). The protein expression of ACC was upregulated in adipose tissues of Adipo-H363Y (Fig. 7A). Further analysis revealed that the total protein level of isoform ACC2, but not ACC1, and that of acetylated ACC2 were augmented significantly in adipose tissues of Adipo-H363Y (Supplementary Fig. 14A). Both SIRT1 and ACC2 were present in mitochondria isolated from the three groups of mice (Supplementary Fig. 14B). Both calorie restriction and biotin-deficient diet decreased the total amount of ACC2 in adipose tissues of Adipo-H363Y (Fig. 7B). The amount of acetylated ACC2 was also reduced by the treatments (Fig. 7C). To confirm that ACC2 is involved in regulating the total amount of biotin, specific chemically modified ACC2 siRNAs were used to treat WT and Adipo-H363Y in vivo. After treatment, epididymal adipose tissues were collected for analysis. Reducing total ACC2 protein levels significantly decreased the amount of biotin in adipose tissues of both types of mice but increased their SIRT1 activities (Fig. 7D). Moreover, ACC2 siRNA treatment significantly inhibited the biotinylation and acetylation of histones in adipose tissues of Adipo-H363Y (Fig. 7E). In addition, recombinant adenovirus–mediated overexpression of hSIRT1(H363Y) in cultured 3T3-L1 adipocytes significantly increased the protein levels of ACC2, the intracellular accumulation of biotin, as well as the amount of biotinylated histones (Fig. 7F).
Histones are the most conserved protein substrates of SIRT1 (26). To define the interrelationships between histone acetylation and biotinylation, a series of histone H3 peptides containing 21 amino acid residues with acetylated K14 and/or biotinylated K18 was synthesized (Supplementary Fig. 15A). In vitro deacetylation assays were performed to compare SIRT1-mediated deacetylation of acetyl-H3 with that of biotinyl-acetyl-H3 (Supplementary Fig. 15B). The ratios of nicotinamide (NAM) to NAD and deacetyl-H3 to acetyl-H3, which were calculated based on the sizes of the peaks, were both significantly reduced (by ∼80%) in reactions containing the peptide with biotinylated K18 (Supplementary Fig. 15B), indicating that biotinylated histones are less susceptible to SIRT1-mediated deacetylation. When biotin was present in the reaction buffer, SIRT1 did not cause deacetylation of acetyl-H3 peptide (Supplementary Fig. 15C). In fact, the addition of biotin facilitated the formation of biotinyl-acetyl-H3 peptide clusters from acetyl-H3 peptide (Supplementary Fig. 15C).
These results demonstrate that upregulated ACC2 in adipose tissues of Adipo-H363Y plays a major role in causing accumulation of biotin, which either directly inhibits the endogenous SIRT1 activity (14) or promotes histone biotinylation, in turn preventing SIRT1-mediated deacetylation.
Adipose tissue stores the surplus of energy as triglycerides and regulates whole-body energy metabolism by secreting adipokines that elicit endocrine/paracrine functions. During aging, the distribution and composition of body fat change significantly (27). A decline of fat storage in adipose depots and an elevation of fat accumulation in ectopic organs (e.g., liver and skeletal muscle) contribute to the deterioration of systemic metabolic functions, including insulin resistance, hyperlipidemia, and hyperglycemia (28). Overflow of lipids into organs other than the fat depots is also a significant contributing factor to the accelerated aging process in obese populations (29). Calorie restriction is the most robust nongenetic approach to extend life span and improve health in mammals (30). It counteracts obesity and related metabolic complications by promoting weight loss and normalizing the function of adipose tissues (31). In particular, calorie restriction increases the production of adiponectin, an insulin-sensitizing adipokine (32), and represses inflammation in adipose tissues (33). Yet, the molecular mechanisms underlying the beneficial effects of calorie restriction are still poorly understood. It has been questioned whether the actual decrease in calorie intake or the restriction of varying nutrients accounts for life span modulation by calorie restriction (34).
The current study demonstrates that restricting certain dietary components, in particular biotin, impacts positively on energy metabolism and prevents metabolic aging, by enhancing SIRT1 activities in adipose tissues. Biotin and its metabolite, biotinyl-5′-AMP, are potent and competitive inhibitors of SIRT1 (14). Biotinyl-5′-AMP occupies the NAD binding site and prevents the breakdown of NAD by SIRT1. Since NAD also acts as a cofactor permitting SIRT1 to interact with protein substrates, inhibition of NAD binding by biotinyl-5′-AMP prevents the interactions between SIRT1 and acetylated protein substrates (14). Biotin occupies the binding pocket of NAM, which may prevent the conformational change from nonproductive to productive SIRT1 (35,36). Calorie restriction reduces total biotin levels in adipose tissues; as a result, the direct inhibitory effects of this vitamin on SIRT1 activity in such tissues of calorie-restricted WT and Adipo-H363Y are alleviated.
The present results further suggest that biotinylation of protein substrates represents an indirect route by which biotin interferes with the enzymatic functions of SIRT1. Biotinylated histone peptides are resistant to SIRT1-mediated deacetylation. Although being relatively rare (37), biotinylated histones have been detected in human cells and play a functional role in gene silencing, responses to DNA damage, and cell proliferation (38). Spontaneous histone biotinylation occurs in the presence of biotinyl-5′-AMP and in the absence of biotin protein ligases (39). Thus, an increased intracellular biotin content and/or turnover can elevate the amount of biotinylated histones. So far, 11 biotinylation sites in histones have been identified, including K9, 13, 125, 127, and 129 in histone H2A; K4, 9, 18, and 23 in histone H3; and K8 and 12 in histone H4 (40). Biotinylated histones contribute to epigenetic modulations involved in the regulation of chromatin structure/stability and genome activation/inactivation. This conclusion is supported by the present observation that different levels of histone biotinylation determine the distinctive patterns of aging-induced chromatin remodeling. Biotin-containing nucleosomes maintain a more condensed average structure (41). In Adipo-H363Y, increased biotinylation prevents endogenous SIRT1-mediated deacetylation of histones and renders the genome more rigid even at a relatively young age. The resulting structural changes alter the chromatin activities of Adipo-H363Y, causing an imbalanced production of pro- and anti-inflammatory adipokines, abnormal expression of genes involved in lipid metabolism, adipogenesis and extracellular matrix deposition, and aberrant genetic adaptations to external conditions and aging. However, calorie restriction can restore the nucleosomal remodeling process and reset the basal state of the genome structures in adipose tissues of Adipo-H363Y. The current study further demonstrates that the beneficial effects of calorie restriction can be mimicked by restriction intake of biotin, which directly (14) and indirectly (by modifying its protein substrates) enhances the endogenous activity of SIRT1.
The initial breakthrough of identification of SIR2 as an enzyme came along with the identification of the Salmonella typhimurium CobB protein, a SIR2 homolog (42). CobB compensates for the lack of CobT mutants during vitamin B12 biosynthesis and possesses nicotinate mononucleotide (NaMN)–dependent phosphoribosyltransferase activity. Thus, CobB catalyzes the release of nicotinic acid from NaMN, whereas SIRT1 removes NAM from NAD. Biotin and NAM were originally discovered as the same class of heat-stable vitamins (43). Unlike NAM, nutritional deficiencies of biotin are rare. Biotin functions in mammals as a CO2 carrier for reactions in which a carboxyl group is transferred to one of four biotin-dependent carboxylases. Consequently, biotin participates as an important cofactor in gluconeogenesis, fatty acid synthesis, and branched-chain amino acid catabolism (44). During aging, the deacetylase activity of SIRT1 is decreasing progressively in adipose tissues, and this is accompanied by excessive biotin accumulation (14). The evidence from the current study and a previous report suggests that biotin supply in diet dominantly inhibits SIRT1 activity and abolishes the beneficial functions of this antiaging protein (14). However, a self-defending mechanism for preventing excess accumulation of biotin is controlled by SIRT1 via ACC2, a major intracellular reservoir and a mitochondrial store of biotin (14,45). The present results suggest that by reducing the total protein amount of ACC2, SIRT1 can regulate the intracellular biotin contents in adipocytes. Under conditions of low biotin intake, enhanced SIRT1 activity downregulates ACC2, leading to a further decrease of intracellular biotin content and the elimination of biotinylated histones, in turn facilitating the dynamic chromatin remodeling and genome activation/inactivation.
The highly conserved H363 residue of SIRT1 forms the binding site for NAD. In yeast, the hSIRT1(H363Y) equivalent mutant SIR2(H364Y) possesses an impaired NAD-dependent histone deacetylase activity (7,26). The strong dominant-negative effects of SIR2(H364Y) have been suggested to result from the incorporation of an inactive SIR2 into silencing complexes. In mammalian cells, whereas the histone deacetylation activity of hSIRT1(H363Y) is impaired, the silencing effects of this mutant are not completely lost (46,47). The hSIRT1(H363Y) mutant exhibits defective substrate binding and catalytic activity that are not locus or species specific (35). The current study reveals that in adipose tissues of Adipo-H363Y, increased biotin contents and augmented biotinylation of histones play a dominant-negative role in preventing endogenous SIRT1-mediated deacetylation and chromatin remodeling. In adipose tissues of Adipo-SIRT1, the interactions between SIRT1 and ACC2 are significantly enhanced and more sensitive to nutritional changes. By contrast, mice with overexpression of hSIRT1(H363Y) in adipose tissues show significantly decreased interactions between ACC2 and SIRT1 (data not shown), leading to a persistent elevation of the protein levels of ACC2, both under basal conditions and during dietary interventions. The present results demonstrate that downregulation of ACC2 (the mitochondrial form of ACC [48]) reduces the biotin content in adipose tissues and leads to a significantly decreased level of histone biotinylation. Previous radioactive tracer studies suggest that a significant portion of biotin is localized as bound forms in both nuclei and mitochondria (49). Whereas ACC2 and SIRT1 are both localized in mitochondria, the amount of SIRT1(H363Y) in this organelle is significantly reduced, which may be attributed to the augmented ACC2 protein levels in adipose tissues of these mice.
In summary, histone biotinylation was significantly enhanced in adipose tissues of Adipo-H363Y, causing deranged epigenetic modifications and chromatin remodeling, which contributed to the accelerated aging phenotype in these mice. Calorie restriction reduces biotinylated histone levels in adipose tissues of Adipo-H363Y, allowing nucleosome deacetylation to occur in these mice at similar levels as in WT. However, unlike biotin-deficient diet, calorie restriction could not further improve most of the metabolic parameters beyond the levels of those in Adipo-SIRT1, which correlates with its milder effects on biotin reduction in mouse adipose tissues. By contrast, the biotin-deficient diet more aggressively decreases biotin contents in adipose tissues of WT and Adipo-H363Y beyond those in Adipo-SIRT1, supporting the interpretation that short-term biotin restriction in the diet is more effective in improving metabolic function than a general restriction of total calorie intake. Thus, although biotinylation directly inhibits SIRT1-mediated deacetylation of histones, calorie restriction or a diet deficient in biotin prevents histone biotinylation independent of SIRT1, which subsequently facilitates gene silencing and prevents aging (Fig. 8). Based on this information, one could speculate that in mammals, caloric restriction may enhance SIRT1 activity by selective depletion of biotin storage in adipose tissue, in turn preventing aging-associated metabolic disorders and promoting life span extension.
Article Information
Funding. This work was supported in part by grants from Seeding Funds for Basic Research of The University of Hong Kong, Research Grant Council grants (HKU779712M and HKU780613M) of Hong Kong, the Special Equipment Grant Scheme (SEGHKU02) from University Grants Committee, HKSAR, and the National Basic Research Program of China (973 Program 2015CB553603).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. C.X. performed the experiments, analyzed the data, presented the results, and prepared the manuscript. Y.C. and P.F. performed the experiments, analyzed the data, and presented the results. B.B. and J.C. performed the experiments. H.-B.D. analyzed the data and presented the results. C.-M.C. obtained funding support and revised the manuscript. A.X. obtained funding support, designed the experiments, supervised the project, and revised the manuscript. P.M.V. obtained funding support, designed the experiments, supervised the project, and prepared and revised the manuscript. Y.W. obtained funding support, designed the experiments, supervised the project, analyzed data and presented the results, and prepared and revised the manuscript. Y.W. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.