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What are the effects of combining rapamycin with dietary restriction?

What are the effects of combining rapamycin with dietary restriction?


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Are the effects additive or subadditive? In many ways, rapamycin acts like a CR mimetic, but even CR can only go so far.


To clarify; administration of rapamycin (a drug) to lab organisms (including mice [1]) extends lifespan. Similarly, restricting the intake of nutrients to the minimum without causing malnutrition also extends lifespan in lab animals (including primates [2]).

Rapamycin inhibits the mTOR pathway (mammalian Target Of Rapamycin) - specifically mTORC1 (Complex 1) - which influences protein synthesis, autophagy and inflammation (among others). Upstream factors of mTOR include nutrient availability and insulin signaling (see "Deconvoluting mTOR biology" for good review [3]).

It has been hypothesized that the lifespan-extending effects of caloric restriction (CR) are mediated by mTOR (one can see why - mTOR is affected by nutrient availability). In fact it may depend on the method of CR;

Greer et al [4] report that different methods of CR in C.elegans, for instance feeding them a diluted food source, or conversely feeding them on alternate days, do not necessarily require the same genetic pathways. Not only this but CR combined with a genetic mutant (eat-2) have additive lifespan-enhancing effects.

So whilst the evidence is not concrete, and I look forward to other studies in mammals similar to the one by Greer et al, it looks as though rapamycin and CR have similar but not exactly the same effects on lifespan; rapamycin specifically inhibits an individual pathway which is involved in many processes, and some of its effects are not necessarily desirable (e.g. rapamycin inhibits the immune system [5]). On the other hand, CR (most of the different types) seems to be mediated by mTOR - this difference is critical: mTOR is not necessarily inhibited by CR, it is just required for its effect.

Therefore combining rapamycin and CR is unlikely to have an additive effect as rapamycin may override any influence CR has on mTOR signaling, but I have not seen a study in which this has been tried. Combing different methods of CR (or developing drugs to do just that) may well have additive lifespan-enhancing effects.

  1. Harrison DE, Strong R, Sharp ZD, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 2009;460(7253):392-5.
  2. Colman RJ, Anderson RM, Johnson SC, et al. Caloric restriction delays disease onset and mortality in rhesus monkeys. Science (New York, N.Y.). 2009;325(5937):201-4.
  3. Weber JD, Gutmann DH. Deconvoluting mTOR biology. Cell cycle (Georgetown, Tex.). 2012;11(2):236-48.
  4. Greer EL, Brunet A. Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C. elegans. Aging cell. 2009;8(2):113-27.
  5. Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nature reviews. Immunology. 2009;9(5):324-37.

This Obscure, Potentially Dangerous Drug Could Stop Aging

Is a rapamycin a miracle drug? Or is it just killing men slowly?

Louis is a 27-year-old assembly-line worker in Three Rivers, Michigan. He has no health problems and rarely sees a doctor. Yet for a man in his prime, Louis thinks a lot about cheating death. He researches strategies online, and he even converted to a plant-based diet after hearing from a YouTube channel called Vegan Gains that veganism could extend his life. Louis thinks the diet will buy him a few extra years, but he feels the urge to keep seeking new life-extending methods. &ldquoI&rsquod like to live as healthily as possible for as long as possible,&rdquo he says. &ldquoAnd if we have therapies and practices today that will prolong our healthy life span, I believe we need to follow these therapies and practices.&rdquo

Charles is your average 50-year-old middle-class family man. He lives in the suburbs of Atlanta, works a 9-to-5 in marketing, does Brazilian jujitsu, and spends weekends watching his kid at wrestling tournaments. Except Charles has a hang-up: He worries about feeling like he&rsquos withering, about those growing-old hallmarks like thinning hair, losing a step, and forgetting his buddy&rsquos wife&rsquos name. Charles is the kind of guy who&rsquos active on antiaging Internet forums and takes fistfuls of supplements. &ldquoA few years back, when my granddad had cancer, I watched him die,&rdquo he says. Charles read up on alternative ways his granddad could attempt to extend his life, but his grandfather didn&rsquot try them. Soon after, Charles (who asked not to use his real name) got some news that sent his hang-up into hyperdrive. &ldquoI took this 23andMe genetic test,&rdquo he says. &ldquoI found out I have a risk for Alzheimer&rsquos.&rdquo It&rsquos a risk that increases as your body ages.

Van is a 72-year-old who managed medical-device sales in Boston until he retired and moved to Spain. He used to run five miles and lift weights three times each per week. But in his late 60s, all that wellness stuff wasn&rsquot working so well. &ldquoI began getting really tired in the afternoons,&rdquo says Van, who also didn&rsquot want to give his name. &ldquoI&rsquod be too tired at night to go out to dinner, and I also started having high blood pressure. I was feeling the effects of aging.&rdquo His attempts at flinging a kettlebell were halfhearted, and his walks around the neighborhood were getting slower. Van felt the sun was setting on the life of vitality he loved and that he was heading toward a gloomy and inevitable bedridden demise.

Each of these men found a solution to their concerns about aging at roughly the same place and time. They came across information in Reddit threads and on longevity blogs about something that people on the other end of the keyboard said could help them live better for longer. It would make Louis healthier now, as he aged, and as all of his friends saw their bodies fade. It would slash Charles&rsquos risk of Alzheimer&rsquos and have guys ten years younger tapping out against him in BJJ. It would kick-start Van&rsquos training again and drop his blood biomarkers to those of someone half his age. It had the potential to be more powerful than diet and exercise. But it also had the potential to cause some problems.

It was a curious substance discovered in soil that had been scooped up on Easter Island during a 1964 Canadian research expedition. Scientists studying disease there noticed that people didn&rsquot pick up tetanus via their feet as they would expect, and they figured the ground held some secrets. But nobody expected to find this one. The soil sat in frozen storage in a University of Montreal lab until 1969, when a researcher looked for useful compounds in it and came across a molecule that was a powerful immunosuppressant. In 1999, the FDA approved the molecule as the drug Rapamune (sirolimus), also known as rapamycin. By the mid-2000s, rapamycin was found to increase the life span of worms and yeast, and in a 2009 study, it extended the life expectancy of mice by 28 percent for males and 38 percent for females. Twenty-eight percent, plus more energy? That could translate into more than a decade of better years for humans, the Redditors and bloggers said. But there was a catch.

Rapamycin wasn&rsquot exactly benign, nor was it something Louis, Charles, or Van could just go pick up at CVS. In high doses, rapamycin suppresses your immune system. The FDA approved it for people who&rsquod undergone organ transplants to keep their bodies from rejecting the donated organ. The stuff could put you at risk of side effects. Roughly 5 percent of patients in clinical trials experienced them badly enough that they had to quit the drug. The FDA stamped rapamycin with a &ldquoblack box&rdquo warning, its most extreme, for drugs that come with &ldquoserious or life-threatening risks&rdquo&mdashrisks like infections, pneumonia, and cancer.

Still, the forums had links to legit research and showed lots of buzz in the ranks of influential biohackers like Tim Ferriss and physicians like Peter Attia, M.D., who&rsquove had MIT researchers and University of Chicago doctors appear on their podcasts talking about the drug&rsquos potentially age-bending benefits. Doctors generally won&rsquot openly prescribe rapamycin for longevity. Van got lucky and found someone who did. But Louis and Charles, like many people who want something today, went looking around the web.

We talked to many men like Louis, Charles, and Van for this story. They range in age from 27 to 76, and their opinions on the drug go from &ldquoprobably helpful but no better than exercise&rdquo to &ldquoeasily the most important drug ever discovered by mankind and should be a key topic of discussion in the upcoming presidential election cycle.&rdquo They are manual laborers, academics, medical doctors, entrepreneurs, and everything in between. There are hundreds, perhaps thousands, of these men quietly experimenting with rapamycin across the country. And if these guys are right, they could be like the lucky rodents in the research, walking around with improved brain health, heart health, and vitality while the rest of us surrender to mortality. Or they could be killing themselves slowly. It&rsquos too soon to tell.

SCIENTISTS STILL DON'T KNOW what actually causes aging. Maybe it&rsquos that your cells stop dividing, or that your telomeres shorten, or that you exhaust your stem cells, or that your DNA becomes damaged and stops repairing itself, or a combination of all those processes. Or maybe it&rsquos none of them. All we can do to live longer and better for now is treat aging&rsquos symptoms. So that&rsquos what the antiaging community until this point has been stuck with trying to do.

It&rsquos a history of whack-a-huckster. In the 1800s, the treatments were patent medicines like Clark Stanley&rsquos Snake Oil Liniment and Hamlin&rsquos Wizard Oil. In the 1920s, antiaging doctors charged $750 to $2,000 for life-extending gland transplants. Medical boards in the late &rsquo30s intervened to cut down on these quack treatments. But in the &rsquo90s, boomers brought the quack back. This generation was hitting middle age and, having grown up in the turbulent &rsquo60s, was willing to question the establishment&mdashin this case, the medical establishment&mdashand turn to self-help. Boomers began popping questionable OTC supplements and getting HGH injections, all in the hope of extra life. In 2002, when the antiaging market hit $43 billion, a group of 51 scientists in the field published a statement in Scientific American decrying the burgeoning, built-on-almost-nothing business of antiaging medicines. No one really cared. Just five years later, the market was expected to reach $64 billion.

Which is about the time Silicon Valley stepped in with big data, big science, and big money to, of course, &ldquodisrupt&rdquo death. Larry Page, Peter Thiel, Jeff Bezos, and other tech billionaires have since funneled billions into life&mdashextension companies such as Calico and the Methuselah Foundation. Much of the new research is founded on an antiaging discovery that occurred all the way back in 1935. It was then that researchers at Cornell found that rats that spent their lives in a state of caloric restriction lived longer.

Eventually, this finding would be connected to that life&mdashextending compound in the Easter Island soil through the microscope of David Sabatini, Ph.D., M.D. Dr. Sabatini didn&rsquot set out to get mixed up in this odd world of antiaging, but in 1992, peering down at a sample he was analyzing one day as a student at Johns Hopkins Medical School, he discovered a protein, now called mTOR (short for mammalian target of rapamycin), that would eventually form a link between the way rapamycin might extend life and the way caloric restriction does. He had discovered the mTOR cellular-signaling pathway that answers to rapamycin. The drug just might act on the very causes of aging.

Dr. Sabatini, now a professor of biology at the Whitehead Institute and MIT, explains the mTOR pathway like this: Pretend your body is an old house. Your oldest cells have all sorts of problems and are implicated in your house falling apart. &ldquoYou couldn&rsquot fully renovate the old house by bringing in only a plumber, or only an electrician, or a roofer, or a drywall guy,&rdquo says Dr. Sabatini. &ldquoYou&rsquod need to hire a general contractor, who would hire all those specialists who would then come fix all those problems that needed to be fixed.&rdquo The mTOR pathway is like the general contractor, signaling to your body to demolish parts of its old cells and replace them with newer, healthier ones.

Dr. Sabatini thinks rapamycin essentially tricks the body into thinking that it&rsquos in a state of calorie deprivation, which is what causes the contractor to call in all the guys for renovation work. The cellular workers consume your oldest, weakest cell parts, even parts of senescent cells. These are cells that no longer divide and are thought to spur aging and maybe even drive cancer. Which is to say that rapamycin could give you all the benefits of fasting without the ravenous downsides. In addition to the studies on rapamycin in yeast, worms, flies, and mice, in 2014 scientists began work on dogs it found that those on the drug showed signs of younger hearts and a reversal of age-related cardiac issues.

While this was going on, Mikhail Blagosklonny, Ph.D., M.D., a prolific researcher on aging at the Roswell Park Comprehensive Cancer Center in Buffalo, began writing about his theories on rapamycin in medical journals. He noted its antiaging promise in 2008 and hypothesized that a lower dose than transplant patients take might bring on benefits without side effects. On Christmas Eve in 2014, a study conducted by researchers at Novartis and Stanford and published in Science Translational Medicine confirmed Dr. Blagosklonny&rsquos theory. Older people taking the drug for six weeks didn&rsquot see their immunity decrease&mdashit actually increased in groups that took as little as 0.5mg a day or 5mg a week. Adult transplant patients typically take a 2mg dose every day.

Today, more than 2,000 clinical trials studying rapamycin are under way around the globe, nearly 1,000 of them in the United States, and even the director of the NIH has blogged about its potential benefits. All of which means rapamycin checks a lot of boxes in the description of a trend that&rsquos about to explode: wellness gurus talking it up, credible researchers giving it ink, and enough unknowns to interpret the research in any way that works with your world-view. But the science, as science does, is proceeding slowly and carefully and may never find answers. Because the drug is already generic, drug companies aren&rsquot interested, and antiaging enthusiasts are going ahead and taking it, sometimes with severe consequences.

IN THE WORLD OF RAPAMYCIN for antiaging, guys find ways to get the drug. Louis and Charles searched the forums&mdashnot the somewhat moderated ones, like Reddit&rsquos, but forums Charles would &ldquorather not mention, just to protect them, you know?&rdquo Charles found a post with a link to an obscure, unregulated pharmacy in India that was willing to ship anyone rapamycin, no prescription needed. Louis got his from a supplier he won&rsquot disclose.

Van located the one doctor in the U.S. who would prescribe the drug. His name is Alan Green, M.D., and he treats patients out of his home in Bayside, Queens. Because you have to visit him in person, Van flew to LaGuardia Airport, took a cab to Bayside, and wound up in Dr. Green&rsquos office.

With these antiaging meds, there are the sketchy, we-don&rsquot-know-the-effects-yet prescriptions&mdashthe ones you get from Dr. Green&mdashand then there are the sketchy sketchy ones from overseas pharmacies and underground suppliers. Many foreign pills are fine, but some aren&rsquot. The bad ones can be counterfeit, contaminated, tainted, or otherwise unsafe. They can make you sick, lead to dangerous interactions with other medicines you take, and even kill you. In Charles&rsquos and Louis&rsquos eyes, the bigger risk was doing nothing. They each asked for a shipment of rapamycin Charles got a four-month supply for $100, and Louis got two years&rsquo worth for about $200.

Van&rsquos prescription came from Dr. Green, who takes the drug himself. The doctor&mdashfive-foot-ten and 175 pounds when he started, with a rim of white hair around his tan head&mdashsays the decision to take it was easy. He had just turned 72, and &ldquoeverything was going to shit. It was clear that I was going downhill fast,&rdquo says the now-76-year-old. &ldquoI would get winded easily and wasn&rsquot moving as well. I knew I wouldn&rsquot be alive much longer if I kept deteriorating at this rate.&rdquo He came upon Dr. Blagosklonny&rsquos work and wrote himself a prescription for 6mg once a week. &ldquoI didn&rsquot have anything to lose,&rdquo he says. &ldquoThe first thing I noticed is that it became easy to lose weight. I was losing two pounds a week. I also had a marked increase in energy and wasn&rsquot getting winded as easily.&rdquo

Dr. Green continued his treatment for a whole year with nothing but upsides. &ldquoSo I decided, &lsquoWell, I&rsquoll make this available to other people,&rsquo&thinsp&rdquo he says. &ldquoThe FDA&rsquos black-box warning is an excellent warning&mdashas it applies for use for organ transplants.&rdquo At the doses for antiaging? Not so much, says Dr. Green. It&rsquos not illegal for him to prescribe the medication. &ldquoOnce the FDA approves a drug, health care providers generally may prescribe it for an unapproved use when they judge that it is medically appropriate for their patient,&rdquo explains Jeremy Kahn, a spokesman for the FDA. Overall, one in five prescriptions today is doled out for off-label use, like the blood-pressure drug Inderal being used for performance anxiety or the antidepressant Zoloft for premature ejaculation.

Dr. Green created a website laying out the research and began hearing from people who&rsquod found him online. &ldquoI figured I&rsquod see a few patients a month,&rdquo he says. His phone started ringing far more than that. &ldquoI think I was Dr. Green&rsquos second patient,&rdquo says Van. The doctor ran Van through a handful of questions about his health history, why he was interested in rapamycin, and what he hoped to get from it. Then he did some basic blood testing, Van says, and wrote him a prescription. Van filled the Rx, dumped six of the aspirin-sized 1mg pills into his hand, and popped them into his mouth. He flew back to Boston, and at home he&rsquod take that same 6mg dose once weekly. &ldquoMy blood work quickly became that of someone 20 to 30 years younger,&rdquo he says.

Seeing Dr. Green isn&rsquot cheap. You&rsquoll have to travel, and he charges $350 for an initial visit and $100 to $200 for follow-ups. Your insurance won&rsquot cover the meds, which cost $75 to $150 a month. Dr. Green now sees about three patients a week, and &ldquomany are doctors, Ph.D.s, and executives,&rdquo he says. But that doesn&rsquot help guys like Charles and Louis.

They received the rapamycin pills a couple weeks after placing their orders. Charles added a 5mg dose in with all the other supplements he takes and washed the mix down with water. Louis did the same with a 7mg dose. Both guys take their pills once a week. &ldquoI dropped 10 or 15 pounds in the three months I&rsquove been taking it,&rdquo says Charles, who admits he has also been eating better. &ldquoI feel less sore, like I&rsquom taking Advil. I was going to quit jujitsu a couple years ago because of my joint pains, and now I&rsquom rolling with 25-year-olds. My hair also seems a lot thicker.&rdquo Louis has been on and off it for almost a year he says he can&rsquot tell if less joint pain and an improved mood are direct results or merely coincidental. He had some mouth sores, indications of a compromised immune system, but they didn&rsquot rattle him&mdashhe thought they, too, might just be a coincidence.

Concerns about infections and worse are one reason more doctors aren&rsquot prescribing rapamycin. Dr. Green says bacterial infections happen in about 5 percent of patients. &ldquoI&rsquove had a few skin and soft-tissue infections, which I treated with antibiotics,&rdquo he says, and he emphasizes that the infections can get bad quickly without antibiotic treatment. &ldquoI have two patients who&rsquove developed pneumonia and required hospital treatment. In both cases, they&rsquod delayed a few days to a week or more in starting antibiotics.&rdquo

&ldquoI think [prescribing rapamycin] is bordering on unethical,&rdquo says Dr. Sabatini, who does not take the drug. &ldquoI think we&rsquore far away from knowing that there are no downsides to long-term use.&rdquo Dr. Attia, the influential 46-year-old physician focused on longevity, says he won&rsquot prescribe it to other people either&mdashat least not yet. &ldquoI take rapamycin myself, so on some level I&rsquove decided it&rsquos a smart option,&rdquo he says. &ldquoBut I haven&rsquot prescribed it to any patients, except one, who is himself a scientist studying rapamycin. And I think that speaks to my desire to better understand the risks, not only of taking too much but also of not taking enough.&rdquo We&rsquoll likely never be able to study directly whether rapamycin really helps humans live longer, says Dr. Attia. It&rsquos too time-consuming and too expensive to do a study like that well. &ldquoWe&rsquoll have to rely on some combination of proxies,&rdquo he says, and scientists are discussing the development of such tests to analyze what kinds of markers could really pin down what&rsquos happening with the drug.

Yet Louis, Charles, and Van aren&rsquot waiting for science to catch up. They&rsquove been on the stuff for as long as two years and don&rsquot plan on stopping. Maybe the three of them, the rest of Dr. Green&rsquos patients, and the untold numbers finagling rapamycin off the Internet will be among the 95 percent of people who see no negative side effects. That&rsquos assuming the pills they get are legit. And maybe they&rsquoll end up outliving us all. Or maybe they&rsquoll find an unknown, unforeseen consequence. Medicine can be a gamble. Some medications sat on shelves for decades before doctors realized they came with harmful long-term side effects and had to be pulled. The painkiller Vioxx, for example, was linked to 27,000 heart attacks and strokes after it was FDA approved, and the acne drug Accutane dramatically increased the risk of miscarriage and severe birth defects in women taking it during pregnancy. Many later sued its maker.

Not that any of those cases will influence the decisions of the antiagers we spoke to. &ldquoI don&rsquot know if it&rsquoll make me live longer, but hopefully I can head off dementia,&rdquo says Charles. &ldquoAnd I feel good, man. So why not?&rdquo


M ETHODS

Animals and Feeding Regiment

Male C57BL/6 mice used in this study were purchased from The Jackson Labs (Bar Harbor, ME). The mice were fed a mouse chow, 7012 Teklad LM-450 (Harlan Laboratories, Madison, WI) until 2 months of age, after which, they were divided into three dietary regimens: ad libitum (AL), where mice were maintained on a commercial mouse chow, Purina Mills Test Diet Control #1810306 (Purina Mills, St Louis, MO) 40% diet restriction (DR), where the mice were fed 60% of the amount of Purina Mills chow eaten by the ad-libitum fed mice at 3 PM each day. Mice given Rapa were fed ad libitum the Purina Mills chow containing 14 ppm of encapsulated rapamycin in the diet as described by Harrison and coworkers (1). Mice were maintained on the three diets for 6 months, until they were 8 months of age. All procedures followed the guidelines approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio. The body weights and body composition (determined by quantitative magnetic resonance [Echo Medial Systems, Houston, TX]) were measured in the groups of mice at 2 (before dietary regimens) and 8 months of age. Mice were euthanized by carbon dioxide and liver tissues were harvested, snap frozen in liquid nitrogen, and stored at �ଌ until used.

Glucose Tolerance Tests and Insulin Tolerance Tests

Both the glucose tolerance test (GTT) and insulin tolerance test (ITT) assays were performed following 16 hours of food deprivation in 8-month-old mice. Food was removed from the AL and Rapa mice immediately after the DR mice had eaten their daily allotment. GTTs were performed with an intraperitoneal injection of glucose (1.5 g/kg body weight) solution in saline into the mice, and blood glucose was measured 0, 15, 30, 60, and 120 minutes after glucose injection. ITTs were performed with an intraperitoneal injection of human recombinant insulin in saline (Novalin Novo Nordisk) at a dose of 0.75 U/kg body weight, and blood glucose was measured 0, 15, 30, and 60 minutes after insulin injection. Blood glucose concentrations were measured using a One-Touch Ultra glucometer. Area under the curve (4) for GTT and ITT was estimated by summing the numerical integration values of successive linear segments of the glucose disposal curve at each set of time points. The data were expressed as mean ± standard error of the mean and analyzed using one-way analysis of variance with the Holm–Sidak post hoc test.

MTOR Signaling and Autophagy Activity

Frozen liver tissue was homogenized with a Dounce homogenizer in ice-cold RadioImmunoPrecipitation Assay buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with inhibitors of protease and phosphatase (Roche, Indiana) on ice. The supernatant was collected after centrifugation at 4ଌ, 12,000g for 10 minutes. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel followed by transfer to nitrocellulose membrane. Target proteins were detected with the following specific monoclonal or polyclonal antibodies: actin, S6, phospho-S6 (Ser235/236), and LC3 (Cell Signaling, Danvers, MA). mTOR signaling was assessed using the ratio of phosopho-s6 levels to total S6 levels. Autophagy activity was measured using the ratio of LC3II/LC3I, and actin was used as a loading control for both. Data were expressed as means ± standard deviation (SD).

GSH and Thioredoxin Redox State

The GSH redox state was determined by measuring the levels of reduced and oxidized GSH in liver collected from the mice. Fifty milligrams of frozen liver was homogenized in 0.5 mL of 5% PCA/0.2 M Boric Acid/10 μM r-EE solution provided by Clinical Biomarkers Lab (Jones Lab, Emory University). Samples were sonicated to breakdown any aggregates and were then spun down and 300 μL of supernatant were transferred to a new tube. Samples were stored at �ଌ and until assayed. The levels of reduced and oxidized GSH were determined using N-dansyl derivatives and high-performance liquid chromatography with fluorescence detection as described by Jones and coworkers (12).

The Thioredoxin1 (Trx1) redox state status was determined in liver tissue by measuring the levels of reduced and oxidized Trx1 with modifications to the methods described in (13,14). Briefly, frozen liver tissue was homogenized in 20 mM Tris pH 8.0, containing 15 mM 4-acetoamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS Molecular Probes, Eugene, OR), supplemented with protease cocktail inhibitor III (Calbiochem, La Jolla, CA). Cytosolic fractions were obtained from homogenates by centrifugation at 16,000g for 15 minutes at 4ଌ. The cytosolic fractions (1 mg/mL) were then incubated in the dark with 15 mM of AMS in 20 mM Tris, pH.8.0 for 3 hours at room temperature. Excess AMS was removed using Microcon YM-3 (Millipore Corporation, Billerica, MA). Reduced and oxidized Trx1 were then separated on a sodium dodecyl sulfate/15% polyacrylamide gel (Bio-Rad, Hercules, CA) under nonreducing conditions. The gel was transferred onto a polyvinylidene fluoride membrane, and proteins were then detected with a specific Trx1 polyclonal antibody obtained from LabFrontier (Seoul, Korea). The intensities of the bands corresponding to the reduced Trx1 (top band) and the oxidized Trx1 (bottom band) were calculated using ImageQuant 5.1 (Molecular Dynamics, Amersham) software.

Quantitative Real-Time PCR

Total RNA was extracted from frozen liver tissues (25 mg) using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and DNA contamination was removed by Turbo free DNase (Ambion Foster City, CA). The RNA yield of each sample was determined spectrophotometrically, assuming that 1 optical density at 260 nm (OD260) unit = 40 mg/L. The quality of total RNA extracted from each sample was monitored by A260:A280 ratio and 1.0% agarose formaldehyde gel electrophoresis.

One microgram of RNA was used to generate complementary DNA using the Retroscript kit (Ambion). All complementary DNAs were diluted to 1, 1:10, 1:100 before being used as a PCR template. Primers were designed using Primer Express (Applied Biosystem, Foster City, CA) and Primer-BLAST (NCBI). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR Master Mix (Applied Biosystem) in a 96-well plate using Gapdh as a housekeeping control with detection by a 7500 Real-Time PCR Detection System (Applied Biosystem). The primer pairs used are described in Supplementary Table 3. Analysis of the qRT-PCR results was done using the Δ㥌T method, and gene products were assayed using agarose gels and dissociation curves.

Statistical Analysis

Unless specified, all data were expressed as mean ± standard error of the mean and were analyzed by one-way analysis of variance with pair-wise comparisons using Turkey’s post hoc test. Statistical significance is indicated by p less than .05.


Material and methods

The IACUC of the Albert Einstein College of Medicine reviewed and approved of this research protocol protocol number 20170408.

Diets

Diets were purchased from Research Diets Inc. The ketogenic diet (KD) composition in calorie percent ratio of carbohydrate/fat/protein was 0.1/89.9/10.0. The standard diet’s (SD) distribution was 80/10/10. Both diets contain the same quality and quantity of mineral and vitamins and other necessary components. The fat content in both diets was from cocoa butter, with 59.7 gm, 32.9 gm and 3.0 gm of saturated (33.2 stearic and 25.4 palmitic), monounsaturated (32.6 oleic) and polyunsaturated fat (2.8% linoleic), respectively, per 100 gm of total fat, with trace amounts of other fats making up about 3% of the remainder. Omega 6 and omega -3 fatty acids contributed 2.8 gm and 0,1 gm, respectively. The KD and SD contain 6.71 and 3.85 calories/gm of energy, respectively. In general, a mouse needs 13.7 to 14.6 calories from their food [19].

Cancer model and treatment

Four-week old, female FVB/N-Tg(MMTV-PyVT)634Mul/J mice were purchased from The Jackson Laboratories. These mice (100%) develop breast tumors spontaneously during their lifetime. The breast tumors can be seen as early as 5 weeks of age. At four months, 80–94% of these mice will have developed lung metastasis [20,21].

Mice were randomly divided into 6 groups of mice, 34 in total, 17 designated to overall SD groups, 17 to KD groups. All mice were were held at the Animal Institute for one week where they were all administered a SD. After an additional week, i.e. at approximately 2 weeks after arrival, or six weeks after birth, animals were returned to the investigator and were immediately assigned to the dietary groups, namely the SD, SD plus rapamycin at 0.4 mg/kg (SDr0.4), SD plus rapamycin at 4 mg/kg (SDr4), KD, KD plus rapamycin at 0.4 mg/kg (KDr0.4), and KD plus rapamycin at 4 mg/kg (KDr4). The number of mice in these groups were 9,3,5,9,4 and 4, respectively. (Variation in expected numbers was unintentional, but resulted from inadvertent deaths of several animals due to a novice animal husbander). At the third week of the special diets, rapamycin was given to mice by oral gavage with a 22-gauge feeding needle at a dose of 0.4 mg/kg or 4 mg/kg daily for 2 weeks. Mice were then maintained on both diets until euthanasia was required.

Housing and husbandry

The Albert Einstein College of Medicine has an AAALAC accredited animal facility with clean barrier housing for mice. Animal caretakers check mice daily and food and water are provided ad libitum except as required in IACUC approved animal protocols. Routine environmental enrichment includes housing of groups of up to five mice per cage with provision of cotton fiber nestlets or small huts. Temperature and humidity is constantly monitored and kept within acceptable ranges (68–72 degrees F and 30–70%). Three veterinarians and 4 veterinary technicians provide oversight and veterinary care. IACUC provides animal welfare oversight. Our mice were housed and husbanded in the institution’s barrier animal facility. This was not secondary to intrinsic immunocompromise, as in nude mice. It was based, rather, on a requirement specific to the Institutional Animal Care and Use Committee (IACUC) of our institution: that all cancer mice, particularly those receiving chemotherapy (which may compromise the immune system secondarily), must be housed in our institutional barrier to reduce the infection rate. As we employed a spontaneous cancer mouse model in which all mice developed breast cancer after 5 weeks of age and some received chemotherapy, the institutional IACUC required barrier housing. Special training of the first author (YZ) was provided in animal handling, anesthesia, tumor measurement, moribund determination, oral gavage, and cardiocentesis.

Blood sampling and analysis

At designated time points, mice were bled from the tail vein with a 21 G injection needle puncture. The peripheral blood drops were used to measure glucose and Beta-hydroxybutyrate (BHB) separately using Keto Mojo, a blood glucose and ketone monitoring system. Each glucose data point is a daily average of three measurements at 3 different time points 9 am, 1 pm, and 5 pm. The Keto Mojo assay has been validated in an independent study by Augusta University [23] of the University of Georgia health system and further confirmed against the Beckman Coulter AU480 Chemistry Analyzer at Biomarker Analytic Research Core of Albert Einstein College of Medicine.

The serum obtained after cardiocentesis at the time of euthanasia (see below) was used to measure insulin level with an ELISA based on chemiluminescence.

Determination of moribund status and humane endpoints

Animals were examined daily (by YZ) for their overall condition and signs of moribund behavior, and weekly to measure tumor volume. Mice were determined to have reached moribund status when they could no longer reach their food and/or when the sum of tumor volume within a mouse exceeded 4 cm 3 . Once mice reached this condition, they were euthanized within four hours which then constituted the duration of the experiment. All (n = 34) animals were euthanized none were euthanized prior to reaching this point. Moribund mice were euthanized in accordance with IACUC recommendations as well as with ARRIVE guidelines for humane endpoints. All animal welfare considerations were taken, including minimization of suffering and distress, including special barrier housing, as described above. They were anesthetized with 2.5% isoflurane, and blood (≥1 ml) was taken by cardiocentesis resulting in immediate death.

Tumor size and survival measurement

Tumor size was measured weekly with calipers (by YZ). Volume was calculated from the longest (L) and shortest (S) dimensions according to . In this spontaneous breast cancer model, each mouse develops multiple tumors. The combined tumor volume represented by the sum of all visible tumor volumes was used as a surrogate measure of the overall tumor growth rate. (This measure does not include additional growth due to metastases). Longevity was determined when a mouse attained a morbid state, characterized by the inability to reach food and water normally, or when the sum of its tumor volume exceeded 4 cm 3 . Moribund mice were euthanized based on these IACUC approved criteria of our institution. The lung and other major organs were resected and weighed, and the lung-to-body weight ratio was calculated. The tissues were fixed with 10% formalin and prepared for later blinded pathological evaluation.

Comparisons between SD vs. KD groups with respect to tumor size, and lung metastasis weight were performed using non-parametric Mann Whitney U tests. Serum measurement of insulin, BHB, and glucose were compared between groups using unpaired Student t-tests, also used for body weight comparisons. Longevity between groups was compared using log-rank testing.

Statistics

Independent variables are between subjects. We used Prism 8 graph and statistical software. The threshold for statistical significance was 0.05 (5% confidence level). The Log-rank (Mantel-Cox) test was used for survival data, and two-tailed t-test or F-test for tumor weight and insulin data because of the normality of distributions with appropriately tight standard deviations. These data were double checked using Mann Whitney U testing. No additional codes were used in the analysis multiple comparisons were compared using ANOVA for body weight only. No data were transformed, outliers were not removed, and there were no missing or excluded data.


Results

KD and SD had similar effects on body weight of mice

Four-week old, female mice were randomly divided into 6 groups of 4-10 mice each. Three groups were assigned to a standard diet (SD) and three to a ketogenic diet. Within each diet group, after 2 weeks, rapamycin was administered by oral gavage with 22 gauge feeding needle at a dose 0.4 mg/kg or 4 mg/kg daily for 2 weeks.

The mice in all SD groups and KD groups (with or without rapamycin) were given the same caloric energy from start point to the end (< 8 weeks). As shown in Fig 1A, the body weights of the mice in KD groups were not significantly different from those in SD groups.

The blood glucose (A), beta hydroxybutyrate (B), and insulin (C) levels. Each glucose data point is a daily average of 3-9 mice, and each mouse was measured 3 times in that particular day at 3 different time points (9 am, 1 pm, and 5 pm). Each beta hydroxybutyrate data point is a daily average of 3-9 mice with single measurement per mouse. The insulin levels were measured when the mice moribund. The serum was used to measure insulin level with an ELISA method. The blue lines or bars represent the data from SD groups. The red lines or bars are the data from KD groups. r0.4 and r4 means rapamycin at the dose 0.4 mg/kg and 4 mg/kg for 2 weeks, respectively.

KD reduced the blood glucose level in mice

Blood serum glucose concentrations in all mouse after one week in all KD groups (KD, KD r0.4, and KD r4), decreasing further after 3 weeks of KD feeding. Blood glucose was significantly lower than that of mice in all SD groups at all time points. Rapamycin at a low dose did not have a clear effect on the glucose level. Higher dose of rapamycin (4 mg/kg) enhanced the glucose levels slightly in both SD r4 and KD r4. At day 57 the mildly elevated glucose ratios of KD r4/KD and SD r4/SD were 158/127 (p = 0.0042) and 227/199 (p = 0.0223), respectively (Figure 1B).

KD increased the blood Beta-hydroxybutyrate level in mice

After 3 weeks, mice in all KD groups showed at least a four-fold elevation of serum BHB concentrations compared with SD mice. These elevations all reached statistical significance (p <005). Rapamycin did not affect the BHB level (Figure 1C).

KD reduced the blood insulin concentration in mice

At study termination (see Methods), we collected the blood from each mouse and measured insulin levels. As Figure 1D demonstrates, the insulin serum concentrations in all SD mice groups were 8 to 20-fold higher than the levels in the respective KD mice groups (p<0.0005). The paired comparison is shown in Table 1.

Blood Insulin Level and Comparison

Serum bicarbonate, sodium, calcium, potassium, creatinine, BUN, were also measured with no significant differences found between KD and SD groups (data not shown). See Fig 1.

KD inhibited tumor growth and prolonged mouse longevity

Mice were fed SD or KD from 6 weeks of age until they reached a moribund condition. All tumor sizes were measured in each mouse weekly from day 1 to day 56, the time period during which no mice had yet reached moribundity. The combined tumor volumes (sum of all measurable tumor volumes in each mouse) of KD mice were smaller than that of SD mice (mean KD 506 mm 3 vs, mean SD 1262 mm 3 , p <0.0001, 2way ANOVA Fig 2A). Rapamycin further enhanced the tumor growth inhibition: the mean combined tumor volume of KD r0.4 and KD r4 versus KD was 359 mm 3 vs. 506 mm 3 (p = 0.0049) and 195 vs. 506 (p < 0.0001), respectively.

Tumor size and survival. Tumor size (A) was measured once a week. Volume (mm 3 ) was calculated as 0.5 L × S 2 (L and S is the longest and shortest dimensions). The sum of all visible tumor volumes in each mouse was used as its tumor volume, and each point represents tumor volumes from 3-9 mice. Longevity (B) was determined to the time a mouse became moribund. The blue and red lines are the data from SD groups KD groups respectively. r0.4 and r4 represents rapamycin at the dose 0.4 mg/kg and 4 mg/kg for 2 weeks, respectively.

As shown in Figure 2B, the median survival of KD mice increased to 78 days as compared to 65 days for SD mice, a 20% increase (p = 0.0002, log rank test). KD, when combined with rapamycin at dose 4 mg/kg further increased the median survival to 95 days when compared to KD diet alone (78 days, as above, p = 0.002) and vs. SD r4 (81 days, p = 0.0049). See Fig 2.

KD reduced metastases in the lungs of mice

Moribund mice were euthanized and their major organs were resected as described in Methods. Only lungs were found to have metastatic tumors among all organ systems. Lungs were weighed before further pathological evaluation. The lung/body weight ratio of each mouse was also calculated as it is positively related to the lung tumor number (n) and/or size [21]. The data is shown in Figure 3A and Table 2.

Lung/Body Weight Ratios (mg/g)

The lung metastases. Moribund mice were weighed, and their lungs were resected and weighed after taking ≥ 1 ml blood out from cardiac puncture. (A) The lung-to-body weight ratio from each group is presented. Round dots represent KD groups. Triangle dots represent SD groups. r0.4 and r4 represent rapamycin at doses of 0.4 mg/kg and 4 mg/kg separately. (B) The lung tissue pathology images are shown. The top row demonstrates lung tissue sections from SD, SDr0.4, and SDr4 groups. The bottom row shows lung tissue sections from KD, KDr0.4, and KDr4. The arrows point out tumor nodules. The magnification is 2.5 × for all pictures. The overall mass of tumors is reduced in the KD images.

There is a significant difference of average lung/body weight ratios between SD and KD groups: 26.0 ± 2.2 mg/g vs. 15.3 ± 3.3 mg/g, p < 0.0001. SD/KD with rapamycin at the lower dose of 0.4 mg/kg trended toward further inhibition of lung metastasis (p=0.0542), with lung/body weight ratio 23.1 ± 3.3 vs. 15.4 ± 4.5 mg/g. Rapamycin at a higher dose (4 mg/kg), when combined with KD showed more significant reduction of lung metastases: SD r4 vs. KD r4 was 16.4 ± 2.5: 10.2± 1.9 mg/g, p = 0.0045, and KD vs. KD r4 was 15.3 ± 3.3: 10.2 ± 1.9, p = 0.0163, respectively. See Fig 3. (above).


D ISCUSSION

As a first attempt to determine whether Rapa and DR affect similar pathways, we compared the effect of DR and Rapa on various pathways that have been proposed to play a role in aging. Starting at 2 months of age, 6 months of feeding Rapa or DR has a similar effect on the mTOR pathway in liver mTOR signaling (measured by phosphorylation of ribosomal protein S6) was reduced approximately 50% (p = .022 Rapa compared with AL and p = .003 DR compared with AL), and autophagy (measured by the LC3II/LC3I ratio), a downstream pathway regulated by mTOR, was increased twofold (p = .035 Rapa compared with AL and p = .024 DR compared with AL). Thus, both DR and Rapa alter the major nutrient sensing system in liver to the same extent. Because the liver is the first organ to receive the nutrients absorbed by the intestine ( 34), these data indicate that at 14 ppm Rapa is equivalent to 40% DR with respect to the organism’s mTOR pathway and nutrient response system.

Although both DR and Rapa reduced nutrient sensing to a similar extent, we found that these two manipulations had quite different effects on the other parameters studied. First, as has consistently been observed, DR resulted in smaller body weight gains compared with AL mice in fact, there was very little growth of the DR mice (<5%) over the 6-month study period. In addition to reduced weight gain, DR also altered body composition it resulted in a major decrease in adiposity, which was observed in epididymal, perirenal, and mesenteric fat depots. In contrast, Rapa had no effect on either the weight of the mice or body fat relative to body weight compared with AL mice. We were somewhat surprised that the Rapa did not have any effect on growth (increase in body weight) of the mice from 2 to 8 months of age because one of the hallmark characteristics of Rapa is the inhibition of growth in cells. Rapa has inhibitory effects on cell proliferation in many cancer cell lines such as B-cell chronic lymphocytic leukemia ( 21) and cervical and ovarian cancer cell lines ( 35). Rapa has been reported to reduce bone growth and weight gain in rats over a period of 2 weeks when Rapa is given by gavage (2.5 mg/kg daily) starting at 3 weeks of age ( 16). It is possible that the lack of an effect of Rapa on growth in our study of mice compared with previous work is due to the age of the animals used (8 weeks vs 3 weeks for the rat study) or the route of administration, daily gavage versus feeding microencapsulated Rapa.

One of the most striking differences between DR and Rapa observed in our study was the effect on insulin sensitivity as measured by glucose and insulin tolerance. Previous studies have shown that DR increases insulin sensitivity in mice ( 36) and rats ( 37). In contrast to AL, Rapa tended to reduce insulin sensitivity (though not significantly), whereas DR significantly increased insulin sensitivity (p < .001 GTT DR compared with AL and p = .022 ITT DR compared with AL). Fraenkel and coworkers ( 38) previously reported that normal and diabetic rats given Rapa (0.2 mg/kg) for 2 days had reduced insulin tolerance, suggesting that Rapa might lead to insulin resistance. Because DR and other long-lived mouse models (Ames dwarf and the growth hormone receptor knockout mice) exhibit an increase in insulin sensitivity, it has been hypothesized that insulin sensitivity plays a major role in life-span extension ( 39). However, the observation that Rapa does not increase insulin sensitivity (and may decrease insulin sensitivity, as shown in rats) demonstrates that the increase in life span of Rapa-fed mice is achieved without an increase in insulin sensitivity.

One of the most well-studied theories of aging is the oxidative stress theory, and data from DR have been important in supporting this theory ( 20). DR mice and rats show reduced oxidative damage to various macromolecules ( 40) and have a reduced cellular environment as shown by the GSH/GSSG ratio ( 23). Rapa has been shown to decrease production of reactive oxygen species in hepatocytes of rats ( 41). After only 6 months on a DR diet, DR-fed mice increased the GSH/GSSG ratio, whereas feeding Rapa had no significant effect. The increase in GSH/GSSG ratio indicates a reduced GSH redox state produced by DR. We also observed no significant changes in the ratio or reduced Trx1 to oxidized Trx1 in either DR- or Rapa-treated mice, which indicates that there were no changes to the Trx1 redox state. These data would suggest that Rapa might not reduce oxidative damage over the life span of the mice as has been shown for DR because a more reduced GSH redox state is correlated to decrease oxidative damage ( 42, 43).

To obtain a broader perspective on the pathways/processes altered by DR and Rapa, we studied the effect of DR and Rapa on the expression of genes involved in the cell cycle and the sirtuin family. DR has been shown to decrease cell proliferation and growth, whereas Rapa has been shown to decrease proliferation in cancer cells. The effect of DR and Rapa on cell cycle gene expression varied depending on the transcript studied, for example, they had a similar effect (upregulation) on mRNAs for cyclin D1and p53 however, only DR samples reach statistical significance. For other side, the effect on mRNAs for p16 and p21 were in the opposite direction. The differences in the effect of DR and Rapa on p21 were particularly striking p21 mRNA levels were increased 3.5-fold by Rapa and reduced approximately threefold in DR. We also observed that the level of p21 protein was significantly increased in the liver of the mice fed Rapa compared with the DR and AL mice. Our data agree with previous reports in lymphocytes where Rapa treatment increased p21 protein levels ( 44), and this may explain how Rapa treatment induces specific G1 cell cycle arrest in many cells ( 42, 43).

The sirtuins play an important role in the regulation of a variety of pathways, ranging from maintenance of genome integrity to metabolism. Of particular, importance to this study are the reports that overexpression of Sir2 extends life span in invertebrates ( 45). However, recent studies call these data into question ( 46). There are seven sirtuins in the mammals of which Sirt1 is the Sir2 homolog in yeast. The current data indicate that the protein levels of many of the sirtuins are altered by DR in tissues of rats and mice, for example, Sirt1 ( 47) and Sirt3 protein levels increased significantly in the liver ( 28), Sirt2 protein levels increased significantly in white adipose tissue and kidney but not the liver and brain ( 48), Sirt4 protein levels appear to be reduced but no calculation was done to show significance or magnitude of the decrease ( 49), Sirt5 protein levels were unchanged ( 30), and Sirt6 protein levels increased in the heart, brain, and white adipose tissue ( 50). Our data show that DR upregulates (two- to threefold for all genes except Sirt5, which was increased approximately 50%) the levels of the mRNA transcript of all seven sirtuin genes in liver. In contrast, feeding Rapa between 2 and 8 months of age had no significant effect on the levels of any of the sirtuin transcripts in the liver.

From an in vivo point of view, rapamycin and DR also have common and different effects, for example, both of them have protective effects against age-related diseases, such as cancer, Alzheimer, and Parkinson diseases, and they also shared side effects, such as wound healing problems and immunosuppressant properties [reviewed in ( 51, 52)]. In this regards, it is important to consider that in clinical treatment rapamycin is not used alone, and therefore, some of these immunosuppressant effects may be attributed to interactions with other drugs. Recent studies performed in mouse models indicate that rapamycin acts as an immune modulator rather than an immune suppressant for example, in mouse studies, rapamycin enhances the efficacy of vaccination and inhibits HIV infection ( 53, 54). However, in contrast to DR, rapamycin treatment extends life span in mice even when this treatment was started late in life ( 52). However, whether rapamycin can be used safety in human aging due to the side effect profile of rapamycin treatment is a major question. For example, rapamycin has been associated with the development of lung toxicity in patients, which can be treated clinically ( 55).

In summary, although 40% DR and 14 ppm Rapa have a similar effect on mTOR signaling in C57BL/6J male mice, their effects on several pathways believed to play a role in aging are quite different. Although these data do not allow us to determine definitively whether DR and Rapa could increase life span through different pathways, these data are the first to show that these two manipulations have different effects on a variety of pathways that have been correlated to increased longevity in mice. Based on these data, we propose that it is likely that DR and Rapa affect life span at least partially through different mechanisms in mice. These data are important because they suggest that a combination of DR and Rapa could lead to an increase in life span in mice even greater that that found in DR alone.


Effect of DR and Rapamycin on Various Metabolic Pathways

Miller et al. ( 18) studied the effect of 5 months of DR (60% AL) or rapamycin (14 ppm) on circulating levels of several endocrine factors in UM-HET3 mice ( 10). It is well established that DR reduces circulating levels of IGF-1 ( 75, 76). Miller et al. ( 18) also observed that DR reduced plasma levels of IGF-1 significantly but rapamycin did not. In addition, Miller et al. ( 18) observed that DR but not rapamycin, increased circulating levels of thyroid hormone T4 and reduced circulating levels of leptin. They also studied the effect of rapamycin or DR on circulating levels of FGF-21, a hormone produced by the liver in response to prolonged fast ( 77). Zhang et al. ( 78) reported that transgenic mice overexpressing FGF-21 live longer. Miller et al. ( 18) reported that DR resulted in a dramatic decrease in plasma FGF-21 levels, while rapamycin either had no effect (males) or significantly increased (females) plasma levels of FGF-21. In contrast, Kuhla et al. ( 79) reported that DR (60% AL) increased plasma levels of FGF-21.

Two groups have compared the effect of DR and rapamycin on liver metabolism. Fok et al. ( 70) evaluated the effect of 6 months of DR (60% AL) or rapamycin (14 ppm) on the liver metabolome by measuring the levels of over 1,000 metabolites in the livers of male C57BL/6 mice. DR significantly altered the levels of 173 metabolites however, rapamycin had no significant effect on any of the metabolites studied. Most of the metabolite pathways that were significantly changed by DR were related to regulation of energy status, for example, amino acid, carbohydrate, lipid, and energy (which included the Krebs cycle and oxidative phosphorylation). Interestingly, when mice were treated with both DR and rapamycin, a significant change was observed in an additional 92 metabolites. Thus, a combination of DR and rapamycin had a greater effect on the liver metabolome than DR alone. In a second report, Yu et al. ( 80) studied the effect of 6 months of DR or rapamycin in same mice on various metabolic pathways in liver. Both DR and rapamycin inhibited lipogenesis and activated lipolysis in liver and increased serum levels of free fatty acids. However, only DR activated β-oxidation, leading to the increased production of ketone bodies by the liver. In contrast, Fang et al. ( 64) reported that 5 months of rapamycin treatment (4mg/kg i.p.) significantly increased total ketone body in plasma of mice with a heterozygous genetic background.

Recently, Choi et al. ( 81) studied the effect of DR and rapamycin on the metabolome of yeast. They found that DR had a greater effect on the yeast metabolome than rapamycin, for example, out of 113 metabolites identified, DR significantly altered the levels of

35% of the metabolites and rapamycin

10% of the metabolites. Less than 20% of the metabolites that changed were the same for the yeast treated with DR or rapamycin. Choi et al. ( 81) also found that DR, but not rapamycin, up-regulated genes for β-oxidation in yeast.

In summary, the current studies comparing the effect of DR or rapamycin on various metabolic pathways show that these two manipulations have quite different effects on most of the pathways currently studied. This is particularly striking in the studies in which the metabolome of yeast or mouse was measured.


Immunopathology

Kiwamu Hyodo , Tetsuro Okuno , in Advances in Virus Research , 2020

3.2 Viral manipulation of cellular phosphorylation dynamics

The target of rapamycin (TOR) protein is a highly conserved serine/threonine kinase that acts as a central hub to integrate nutrient and environmental signals ( Xiong and Sheen, 2015 ). TOR regulates various cellular processes, including mRNA translation, energy production, metabolism, and autophagy. Several lines of evidence have suggested the link between plant virus infection and TOR. For example, the CaMV P6/TAV (transactivator/viroplasmin) protein binds to TOR and mediates its activation, leading to the phosphorylation of the ribosomal protein S6 kinase 1 (S6K1) ( Schepetilnikov et al., 2011 ). The phosphorylated S6K1 then activates host translation factors, including the re-initiation supporting protein and subunit h of eIF3, to facilitate the translation re-initiation of viral pregenomic RNA ( Schepetilnikov et al., 2011 ). P6-mediated activation of TOR also results in the attenuation of autophagy induced by the plant defense-related phytohormone, SA, which would be beneficial for CaMV ( Zvereva et al., 2016 ). Downregulation or pharmacological inhibition of TOR attenuates infections by two unrelated plant viruses, CNV and watermelon mosaic virus (WMV genus: Potyvirus family: Potyviridae) ( Inaba and Nagy, 2018 Ouibrahim et al., 2015 ), further supporting the proviral function of TOR in plant virus infection. Notably, CNV infection leads to partial impairment of cellular TOR activity, likely because of the reduced cytosolic ATP concentrations caused by the hijacking of host glycolytic enzymes during virus replication (see Section 4 ), although such mild inhibition of TOR activity appears to not be adverse for virus replication ( Inaba and Nagy, 2018 ). As TOR plays versatile roles in various aspects of the life cycle of plants, it would be interesting to investigate whether virus-mediated alternations of the TOR activity exert some effects on the physiological changes in plant cells caused by plant virus infections.

Phosphorylation dynamics are crucial for the regulation of plant immune responses to bacterial and fungal pathogens ( Withers and Dong, 2017 ). Among them, the mitogen-activated protein kinase (MAPK)-mediated phosphorylation cascades play a central part in the transduction of immune signaling that mediates transcriptional reprogramming for defense ( Meng and Zhang, 2013 ). Although a previous report showed the activation of MAPKs in an incompatible TMV–tobacco interaction ( Zhang and Klessig, 1998 ), our understanding of their roles in plant virus infection is just beginning to unravel. In the case of BaMV, MAPK phosphatase 1 (MKP1) has been detected in an affinity-purified fraction of REPBaMV in N. benthamiana ( Lee et al., 2016 ). The downregulation of MKP1, which dephosphorylates MAPK to downregulate its activity, enhances BaMV and related FoMV replication in N. benthamiana, while the overexpression of MKP1 inhibits viral infection in a phosphatase-activity-dependent manner ( Lee et al., 2018 ). The proposed function of MKP1 in BaMV and FoMV replication is the downregulation of viral RNA replication by attenuating MAPK activity i.e., the activation of MAPK signaling might be beneficial for viral replication. The manner in which MKP1 (and possibly MAPK) is involved in BaMV replication and whether BaMV modulates MAPK activity through MKP1 warrant further investigation. Conversely, MAPK signaling appears to be important for antiviral defense responses to TYLCCNV ( Hu et al., 2019 ). A. thaliana and N. benthamiana can trigger MAPK activation in response to TYLCCNV infection through an unknown mechanism, and loss of MPK4 (a MAPK isoform) or its upstream kinase, MKK2, leads to enhanced viral infection and severe symptoms, supporting the antiviral role of the MKK2–MPK4 branch. Importantly, the βC1 protein of the TYLCCNV betasatellite binds directly to both MKK2 and MPK4 and inhibits their kinase activities, to attenuate the MKK2–MPK4-mediated plant defense response. These two examples suggest a dual role for MAPK signaling in virus replication and plant defense, although further studies are needed to elucidate the exact role(s) of MAPK signaling in various plant–virus interactions.


Rapamycin and Metformin Could Effectively Boost Your Lifespan

For hundreds of years, we have been trying to lengthen our lifespan. Some people have even gone to great lengths to find a source of immortality. A Spanish conquistador named Ponce de Leon spent decades sailing the world in search of the fountain of youth. Unfortunately, he died before he could find it. Ever since the days of his exploits, we have been obsessed with finding a way to make our lives much longer. We have struggled to defeat mortality and now, we could be on the verge of a victory. There are two anti-aging pills named Rapamycin and Metformin. Both are approved by the Food and Drug Administration (FDA). According to scientific testing, they have showed great promise in extending the lives of test subjects. Read on to find out more about the two most promising anti-aging pills of the century.

What is Rapamycin?

In the early 1980s, Dr. Surendra Sehgal discovered Rapamycin in a remote location known as Easter Island. Its traditional name is Rapa Nui. Thus, the doctor derived the drug’s name from it. He actually dug it up from the soil under one of the legendary Moai stone heads on the island. These were created between the 10th and 16th centuries by the inhabitants of the island. Dr. Surendra found a bacterium that was named Streptomyces hygroscopius and its secretion was the basis for Rapamycin.

Early research on the drug and FDA approval

Dr. Surendra performed most of the research on Rapamycin with resources provided by Wyeth Healthcare Company. Wyeth bought the company where the doctor was working at in 1987. As a matter of fact, the doctor made his most significant progress while under Wyeth. In their laboratories, he discovered that the drug could suppress the immune system. Thus, it was approved by the FDA as a treatment that is administered to patients of organ transplant in the year 1999. The doctor died a few years after this approval was granted. As such, he was not able to live long enough to see his discovery ensure that hundreds of thousands of patients stayed alive after transplant. He also did not get to benefit from the drug’s profitability. Wyeth Health Company made hundreds of millions with Rapamycin.

What does Rapamycin do?

Rapamycin suppresses the immune system’s urges to reject new organs. This allows for gradual acceptance of these organs into the body. Today, scientists are beginning to discover that Rapamycin has the ability to delay the development of diseases which are associated with aging. Examples of these include cancer, Alzheimer’s disease and heart disease too. Tests conducted on monkeys, mice and worms have been successful so far.

Its discoverer Dr. Surendra also found that Rapamycin has some anti-fungal properties. Soon after discovering it in Easter Island, the doctor synthesized it into a cream. His neighbor’s wife developed a fungal skin condition and he administered it to her. Amazingly, the rapamcyin worked and his discovery was confirmed.

Rapamycin works at the foundation level of human cell biology. At this level, it acts as a signaler for the metabolic system. This means that the drug can inhibit the biological pathway which is found in human beings. This is the pathway which senses that food has been consumed and there is enough energy for processes such as cell replication, synthesis of proteins and overall growth too. By inhibiting this pathway, Rapamycin is able to prevent expansion of cells in the body when an organism has low energy supplies. This form of dietary restriction increases the lifespan of an organism by between 25% and 30%. When applied on a human perspective, eating fewer calories increases one’s lifespan. This has been observed and documented in some Asian countries where calorie consumption per individual is low. These observations were made by the National Institute of Health as well as a team of scientists from various academic and scientific institutions.

Could Rapamycin be the key to living longer? Well, it could be. But there is one major side effect. During testing, scientists discovered that Rapamycin caused the body to become insensitive to glucose. This could lead to a person developing diabetes. However, further research found the solution to this side effect in another drug, Metformin.

What is Metformin?

This is a drug that works to reduce the effects of diabetes in the body. It makes the liver produce less glucose and increases the sensitivity towards insulin in the body. Therefore, Metformin can balance out Rapamycin in this respect. Metformin also causes the intestines to absorb less sugar. Metformin has been used in Europe ever since 1957. In the United States, it was approved in 1995. It is a safe drug that works to increase sensitivity in diabetics who have weak kidney and liver functions. In addition to that, the drug also has an effect on the development of conditions and diseases which are caused by age. Examples of these are oxidative stress, apoptosis, diminished autophagy and cell senescence. Due to these effects, Metformin got a go ahead from the FDA to be used outside the sector of diabetes. Now, scientists are proposing the idea of using it alongside Rapamycin to see if the combination of drugs can actually extend life.

How can Rapamycin and Metformin increase the length and quality of life?

If the tests are all successful, a combination of Metformin and Rapamycin can be used to treat diseases and conditions that arise due to age. Rapamycin has already been documented to be extremely effective in treating and preventing these conditions. Its side effects are counteracted by Metformin and thus the combination results in safe healthy development. By healing the age-related problems, this combination of pills can improve one’s health during the sunset years and effectively increase the length and quality of human life.

The Important Take Away

For a long time, we have wanted to live for much longer than we currently do. This struggle with mortality has led to some ingenious ideas and products. However, none have ever come as close as Rapamycin in ensuring longer, healthier, more fulfilling life. Dr. Surendra’s discovery could change the course of humanity’s struggle with mortality. A combination of Rapamycin and Metformin could pass the testing period and literally become the fountain of youth.


Lifespan.io Starting Rapamycin Antiaging Human Trials

Rapamycin has been proven to extend the lifespan of mice, warms and yeast. Lifespan.io is starting a large clinical trial named Participatory Evaluation (of) Aging (with) Rapamycin (for) Longevity Study, or PEARL, to see if the antiaging effects of Rapamycin apply to humans. This will be the first study to see if Rapamycin works as well in humans as it does in mice.

The PEARL trial will follow up to 200 participants over 12 months testing four different Rapamycin dosing regimens. It will be double-blind, randomized, placebo-controlled and registered with clinicaltrials.gov. The principal investigator is Dr. James P Watson at UCLA, who was also a PI for the famous TRIIM trial.

Tests and measurements will be taken, both after 6 and 12 months. These will include autonomic health tests, blood tests, body composition tests, fecal microbiome testing, immune and inflammation health tests, methylation age clock testing and skeletal muscle tests.

The current data show that dietary restriction and rapamycin have different effects on many pathways and molecular processes. In addition, these interventions affect the lifespan of many genetically manipulated mouse models differently. In other words, while dietary restriction and rapamycin may have similar effects on some pathways and processes overall, they affect many pathways/processes quite differently. Therefore, rapamycin is likely not a true dietary restriction mimetic. Rather dietary restriction and rapamycin appear to be increasing lifespan and retarding aging largely through different mechanisms/pathways, suggesting that a combination of dietary restriction and rapamycin will have a greater effect on lifespan than either manipulation alone.

He said the following about rapamycin:
If used properly, rapamycin is not much more dangerous than ordinary aspirin. Aspirin, one of the most widely used nonprescription medications, may cause numerous side effects, including life threatening gastric bleeding. The manufacturer lists as possible side effects: ringing in ears, confusion, hallucinations, seizure, severe nausea, vomiting, bloody stools, coughing up blood, fever and swelling. Still, millions of people take aspirin daily to prevent cardiovascular disease and cancer. It was calculated that the benefits of aspirin are greater than their risks. I believe the benefits of the anti-aging effects of rapamycin/everolimus may even be greater.

Rapamycin known in the clinic as Rapamune or Sirolimus, was unlucky from the start, however. Twenty years ago, it was labeled an immunosuppressant and used to treat renal transplant patients. If rapamycin had been labeled an immunomodulator and anti-inflammatory drug instead, it would sound much more appealing now. At anti-aging doses, rapamycin “eliminates hyperimmunity rather than suppresses immunity” or, more figuratively, it “rejuvenates immunity”. This enables rapamycin and everolimus, a rapamycin analog, to act as immunostimulators, improving immunity in cancer patients and the elderly. For example, rapamycin reduces the risk of CMV infection in organ transplant patients, improves antipathogen and anticancer immunity in mice, prolongs lifespan in infection-prone mice and protects aged mice against pneumonia. Rapamycin also inhibits viral replication. As a noteworthy example, rapamycin inhibits replication of the 1918 flu virus (the deadliest flu virus in history) by 100-fold, and also protects against lethal infection with influenza virus when administered during vaccination. Still, as Dr. Allan Green advises, patients taking rapamycin should be carefully monitored for skin and subcutaneous bacterial infections, which should be treated with antibiotics.

Rapamycin and everolimus are FDA-approved drugs, safe for human use. Since 1999, rapamycin has been used by millions of patients with no unexpected problems. One may suggest that rapamycin/everolimus are safe enough for very sick patients, not for healthy people.

Rapamycin reduces the risk of cancer and can extend the lives of short-lived animals by 7-14%.

SOURCES – Lifespan.io, Aging journal, Afar
Written By Brian Wang, Nextbigfuture.com

Brian Wang is a Futurist Thought Leader and a popular Science blogger with 1 million readers per month. His blog Nextbigfuture.com is ranked #1 Science News Blog. It covers many disruptive technology and trends including Space, Robotics, Artificial Intelligence, Medicine, Anti-aging Biotechnology, and Nanotechnology.

Known for identifying cutting edge technologies, he is currently a Co-Founder of a startup and fundraiser for high potential early-stage companies. He is the Head of Research for Allocations for deep technology investments and an Angel Investor at Space Angels.

A frequent speaker at corporations, he has been a TEDx speaker, a Singularity University speaker and guest at numerous interviews for radio and podcasts. He is open to public speaking and advising engagements.


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