Opportunity is missed by most people because it is dressed in overalls and looks like work.— Thomas Edison (from Dr. Julian Whitaker’s
“Health & Healing”, March 2010)Yet, Freedom! yet thy banner, torn, but flying,
Streams like the thunder-storm against the wind.— Lord George Gordon Byron
The pen is mightier than the sword...[...]
only if the sword is very small and the pen is very sharp.— Terry Pratchett, “The Light Fantastic” (1986)
One night I was sitting with friends at a table in a crowded Key West bar. At a nearby table, there was a mildly drunk woman with a very drunk husband. Presently, the woman approached us and asked me to sign a paper napkin. All this seemed to anger her husband; he staggered over to the table, and after unzipping his trousers and hauling out his equipment, said, “Since you’re autographing things, why don’t you autograph this?” The tables surrounding us had grown silent, so a great many people heard my reply, which was: “I don’t know if I can autograph it, but perhaps I can initial it.”— Truman Capote
In my many years I have come to a conclusion that one useless man is a shame, two is a law firm and three or more is a congress.— John Adams
As we have written before, eating a meal, especially one which contains large amounts of calories, fat, and/or carbohydrates, causes the release of inflammatory agents such as ROS (reactive oxygen species), inflammatory cytokines (such as IL-8), master regulator of inflammation NFkappaB,1-2 metalloproteinases, and toll-like receptors (TLR2, TLR4)1 as well as microbial endotoxins (LPS(1,3,4), lipopolysaccharides). As explained in paper #1, “MNC’s [circulating mononuclear cells] constitute the major cellular group (monocytes and T and B lymphocytes) that participate in intramural atherosclerotic inflammation and are known to be in a proinflammatory state in obese individuals who carry a high risk of atherogenesis and have a chronically elevated food intake. Furthermore, inflammatory factors contribute to interference with insulin signal transduction and insulin resistance.” For instance, a recent paper5 reports that TLR2 (Toll-like receptor 2) is critical for diet-induced metabolic syndrome in mice.
A new paper1 reveals in a human study that the MNC’s of subjects who drank orange juice (300 kcal or a little less than 3 cups (110 kcal/cup) of “Not From Concentrate” Florida Orange Juice) along with a 900 kcal high fat, high carbohydrate meal did not experience the meal-induced oxidative and inflammatory stress (such as increased endotoxins and Toll-like receptor expression) that took place in the MNC’s of subjects eating the same meal but along with either 300 kcal of glucose or with water.
The constituents of orange juice responsible for this remarkable protective effect were not determined in this study; however, the authors suggest that the effects are “probably attributable to its flavonoids, naringenin, and hesperidin because they exert a significant ROS [reactive oxygen species] suppressive effect in vitro at concentrations of 50 umol/L. These concentrations are consistent with a flavonoid content of 5-10 mg/100 mL in the consumed orange juice in our studies, assuming a complete absorption from the gut and its distribution in 5.0 L.” Suggestion: Adding 300 kcal (the energy content of the orange juice) to a meal is a lot of additional calories of which the sugar content of orange juice is significant. Yet, it seems very unlikely to us that the sugars could have contributed to the antiinflammatory effect of the juice. Hence, a reduced sugar content orange juice containing the same amount of orange juice constituents as the full-sugar variety should be as effective. Also, note that in this study, the subjects were three groups of healthy, normal weight men and women with BMI of 20-25 and aged 20-40 years. The effect of the sugars in orange juice might not be as benign in, say, obese type 2 diabetics; therefore, such individuals should test their blood sugar changes if trying the orange juice therapy.
Elastase is a proteolytic enzyme involved in the degradation of the extracellular matrix (ECM), that includes elastin. Elastin provides much of the elastic recoil properties of skin, arteries, lungs, and ligaments.1 Loss of elastin is a major part of what causes visible signs of aging (wrinkles, sagging) in skin. Eighty percent of the dry weight of skin is reported to be collagen,1 responsible for the tensile strength of skin. Collagenases are a type of metalloproteinase that can cleave molecules in the ECM that includes elastin, fibronectin, laminin, and collagen. As the authors of paper #1 point out, you need elastase to degrade proteins that are, for example, within the ECM after wounding in order to eliminate this proteinaceous material by phagocytosis to permit repair. However, natural materials with anti-elastase and anti-collagenase properties can help prevent the undesirable age-associated destruction of elastin and collagen.
In a recent study,1 the anti-elastase, anti-collagenase, and anti-superoxide activity of 21 plant extracts were determined in a chemical assay. Nine of the plant extracts exhibited anti-elastase activity, with the highest six being white tea (~89%), cleavers (~58%), burdock root (~51%), bladderwrack (~50%), anise and engelica (~32%). Anti-collagenase activity was exhibited by 16 of the plant extracts, with the highest inhibition being exhibited by white tea (~87%), green tea (~47%) rose tincture (~41%), and lavender (~31%). Nine of the extracts had activity against both elastase and collagenase, as follows: white tea (E:89%,C:87%) > bladderwrack (E:50%,C:25%) > cleavers (E:58%,C:7%) > rose tincture (E:22%,C:41%) > green tea (E:10%,C:47%), followed by rose aqueous > angelica > anise > pomegranate (E:15%,C:11%).
The phenolic content of the extracts varied between 0.05 and 0.26 mg gallic acid equivalents/ml. with the exception of white tea, reported as 0.77 mg gallic acid equivalents/ml.1
White tea was clearly the superior plant extract in this study for anti-elastase and anti-collagenase activity (white tea and green tea also had about the same and the highest level of superoxide scavenging activity compared to the other extracts). The authors also cite a separate study done by other researchers in which 150 plant extracts were tested for their anti-elastase activity, with six inhibiting elastase more than 65% that included cinnamon, turmeric, and nutmeg.
The data speak for themselves. Though chemical assays may differ from in vivo results due to the compartmentalization of chemical systems within cells, the strong anti-elastase and anti-collagenase and anti-superoxide activity of some of these extracts, particularly white tea, along with the safety of drinking white tea (which is just green tea picked at an early phase in its growth) suggests one easy method for protecting elastic tissues, including skin, at least in part, from an important aspect of aging.
A very interesting paper appeared in the March 2010 Nat Biotechnol.1a In ischemic diseases, such as stroke, angina, heart attacks, and sleep apnea, cells are exposed to hypoxia (deficiency of oxygen) with resulting adaptations, such as the induction of HIF (hypoxia inducible factor). One method that cells use to adapt to oxygen or nutrient deficiency is to shift energy metabolism from mitochondrial oxidative phosphorylation (the efficient ATP-generating pathway in which glucose is metabolized to CO2 and water) to glycolysis (a less efficient pathway for generating ATP in which glucose is metabolized to lactate under conditions of hypoxia). “... studies in animal models have shown that attenuating mitochondrial respiration can prevent the pathological consequences of ischemia-reperfusion injury in myocardial infarction and stroke.”1a It has also been suggested that, because mitochondrial respiration is the major source of reactive oxygen species, glucose to lactate (glycolysis) conversion may protect cells that depend upon it (such as rapidly reproducing cells and cancer cells) from oxidative stress.1b
The authors devised a small-molecule screen for detecting agents that shift energy metabolism from mitochondrial respiration to glycolysis in fibroblasts. They were particularly interested in molecules that induced very small shifts in metabolism, as “they may represent particularly safe drugs with which to manipulate energy metabolism.” As a result of this screen, the authors identified meclizine (Antivert), “which has been approved for the treatment of nausea and vertigo for decades, is available over the counter, has a favorable safety profile, and likely penetrates the blood-brain barrier given its efficacy in disorders of the central nervous system.”
Meclizine, they found, acts as a mild uncoupler of cellular oxygen consumption, without an effect on HIF. The researchers tested meclizine in an adult rat ventricular cardiomyocyte model of simulated ischemia-reperfusion injury and found that a 20 minute meclizine pre-incubation followed by washout before ischemia resulted in a dose-dependent protection of cardiomyocytes against cell death. “As with other cell types, meclizine inhibited oxygen consumption in cardiomyocytes in a dose-dependent manner.” The authors also found meclizine to preserve heart pump function after an ischemic event in perfused rat hearts. In another paper,2 the author suggests that “... persistent glycolysis is deleterious due to the generation of MG [methylglyoxal, a metabolite of glucose, and a potent glycating agent*], but brief periods of glycolysis could be hormetic.” In other words, brief periods of glycolysis induced by a mild mitochondrial uncoupler could act similarly to ischemic preconditioning. This might suggest using a mild uncoupler like meclizine at bedtime for people who suffer from intermittent hypoxia during sleep (sleep apnea).
*Our formulation AGEless™ contains potent antiglycating agents (including carnosine, benfotiamine, histidine, alpha lipoic acid, vitamin B6, and rutin) that help prevent deleterious effects of glycating agents such as methylglyoxal.
As the authors explain, the potency of meclizine in attenuating mitochondrial respiration appears to vary across different cell types and, moreover, it is not clear whether the currently approved doses of meclizine (for nausea and vertigo) would achieve dose levels adequate for cardioprotection or neuroprotection. Hence, they suggest that more research be done on optimal dosing for efficacy and safety. However, it is unlikely that there will be much (if any) funding available for such research (though we would love to see it) on a 40 year old drug long beyond patent protection.
The use of meclizine at its current approved doses has a 40 year record of safety; hence, for those with ischemic diseases who are interested in experimenting with this drug, we would suggest you first discuss this with your physician and, if you do decide to use it, do NOT exceed the currently approved dose. If you have cancer, it would probably be a good idea to avoid any substance that would induce continuous glycolysis, as many cancer cells rely on glycolysis (called the Warburg effect in cancer cells) for generating energy, though it is not clear how a substance that induced short periods of glycolysis would affect glycolysis-addicted cancers. We do not recommend experimenting with meclizine if you have cancer.
1a. Gohil et al. Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis. Nat Biotechnol 28(3):249-55 (2010).
1b. Brand and Hermifisse. Aerobic glycolysis by proliferating cells, a protective strategy against reactive oxygen species. FASEB J 11:388-95 (1997).
2. Hipkiss. On the mechanisms of ageing suppression by dietary restriction — is persistent glycolysis the problem? Mech Ageing Dev 127:8-15 (2006).
A new paper1 reports on how NFkappaB signaling plays an important role in aging by its regulatory function in apoptosis, autophagy, and tissue atrophy, and how chronic activation of NF-kappaB can induce senescence.
As reported in paper #1, a meta-analysis of age-related expression profiles of 27 datasets from mice, rats, and humans, the most common age-related genetic signature involved the overexpression of inflammation and immune response genes and also genes associated with the lysosomal system2 (which breaks down cell garbage, as in autophagy). This combination of age-associated changes is often called inflamm-aging. An earlier study by the authors of paper #1 reported that the DNA-binding capacity of the NFkappaB complex was significantly increased in all old rat and mouse tissues they studied as compared to young rat and mouse tissues. “This age-related constitutive activation of NF-kappaB system has been verified later by several other research groups ...”1
“The NFkappaB system is a cytoplasmic sensor in particular in immune attacks but also to a wide array of external and internal danger signals such as oxidative stress, hypoxia, and genotoxic stress.”1
Chronic activation NFkappaB is importantly involved in loss of muscle mass as occurs in cachexia and in age-associated muscle loss (sarcopenia). “Recent studies have convincingly demonstrated that the NFkappaB pathway is the major signaling mechanism, which can induce muscle atrophy.”1 Moreover, NFkappaB signaling potently inhibits autophagy by activating the mTOR/Raptor complex in response to TNFalpha (tumor necrosis factor alpha) and exposure to insulin.1 Conversely, SIRT1 (induced, for example, by caloric restriction or by resveratrol) and FoxO3, two longevity factors that inhibit NFkappaB signaling, powerfully enhance autophagy. Resveratrol and curcumin also inhibit NFkappaB signaling. As the authors1 note, it would be harmful to inhibit the entire NFkappaB signaling pathway, as they illustrate with thalidomide, an NFkappaB inhibitor that was associated with severe birth defects. Hence, the safe NFkappaB inhibitors only moderately reduce NFkappaB signaling.
Other natural substances that inhibit NFkappaB expression include, for example, quercetin,2 lutein,3 EGCG,4 cinnamaldehyde (the major constituent of cinnamon bark oil)5 and alpha lipoic acid.6 On the other hand, high glycemic index carbohydrate (bread, glucose) were reported to increase NFkappaB activation in mononuclear cells of young, lean healthy subjects.7
It is also interesting to note that NFkappaB has been found to be a critical mediator of stress-impaired neurogenesis and depressive behavior,8 thus linking inflammation in the brain to cognitive and emotion regulation dysfunction. The authors of paper #8 report that these findings are consistent with human studies, which show increased NFkappaB signaling in social stress/anxiety.
Inhibitors of mTOR Revisited: Resveratrol
In our Feb. 2010 issue of the Durk Pearson & Sandy Shaw Life Extension News, we discussed (“Life Extension by Inhibiting Growth”) the possible life extending effects of inhibiting the nutrient-sensitive growth-inducing gene mTOR (mammalian Target of Rapamycin). We discussed two potential nutrient inhibitors of mTOR: curcumin and resveratrol. The data we had at that time made it impossible to determine what the effective dose of resveratrol would be for the purpose of effectively and safely inhibiting mTOR. A new paper (thanks, Will) helps to close in on an appropriate dose.
Autophagy and Resveratrol
In the new paper,1 resveratrol was studied for its potential effects on autophagy, a self-digestion process by which a cells’ own components are degraded by lysosomes and the resulting chemicals reused. In a review on autophagy,2 the author wrote: “Autophagy, a highly conserved mechanism of quality control inside cells, is essential for the maintenance of cellular homeostasis and for the orchestration of an efficient cellular response to stress. The decrease in autophagic activity observed in almost all cells and tissues as organisms age was proposed to contribute in different aspects of the aging phenotype and to the aggravation of detrimental age-related diseases.” In another paper,3 the authors write: “... even on a day-to-day basis, autophagy is activated between meals in organs such as the liver to maintain its metabolic functions, supplying amino acids and energy through catabolism.” Increased autophagy is observed in caloric restriction models of lifespan increase in model animals. Other papers4 report that loss of autophagy in the central nervous system causes neurodegeneration in mice by preventing the degradation of inclusion bodies. Moreover, autophagy was reported to be required during cycling hypoxia to reduce ROS (reactive oxygen species) by removing ROS-producing species,5 which may be one reason why inhibiting autophagy increases cancer cell death.
Interestingly, increases in mTOR (as occurs after a meal) causes a decrease in autophagy. Inhibition of TOR by rapamycin induces autophagy. Inhibiting mTOR, then, is one way to increase autophagy.
Resveratrol, mTOR, and Autophagy
In the new paper,1 resveratrol at low doses (0.1 and 1 uM in H9c2 myoblast cells and 2.5 mg/kg/day in rats) induced cardiac autophagy. Autophagy was suppressed, however, by the higher dose of resveratrol (100 uM in the myoblast cells and 25 or 100 mg/kg/day in rats). Treatment with rapamycin, a known inducer of autophagy, did not further increase autophagy compared with resveratrol (low dose) alone.1 It was found that resveratrol attenuated the activation of mTOR complex 1, but significantly induced the expression of mTOR complex 2.
In summation, low dose resveratrol induced autophagy in cardiac myoblasts, as well as increasing cell survival. The effective dose in rats was 2.5 mg/kg/day, the equivalent of about 40 mg/day of resveratrol in humans.
Resveratrol and Polyamines Induce Autophagy
In another recent study,6 resveratrol (and, also, spermidine, a polyamine found in beans, especially soybeans7) were shown to induce pharmacological lifespan extension in model organisms (yeast, C. elegans) via increasing autophagy. Polyamines have also been found to increase lifespan in mice. For example, a recent paper7 reports that orally feeding polyamine-rich food (e.g., found in beans; soybeans were used in this study) for 88 weeks to mice resulted in a significantly lower incidence of senescent progression of glomerulosclerosis in the kidney, as well as increased concentrations of SMP-30, a liver protein that seems to protect organs from oxidative stress during aging.8 In addition, the mice fed the high polyamine containing chow lived longer and had less age-associated pathology and also had thick and healthy fur (as compared to the low polyamine containing chow fed mice) and appeared more active.
Another paper8 on polyamines (spermidine was used in this study) and autophagy reported enhanced autophagy and enhanced longevity in yeast, flies, worms, and human cells. It is important to note that, like other foods and food constituents, you can get different effects at very high concentrations as compared to moderate concentrations.9 It is probably best to get your polyamines from food. As reported in a paper9 on the effects of polyamine breakdown products on inflammation, while the “polyamine spermine has been shown to inhibit pro-inflammatory cytokine synthesis in human mononuclear cells,” “polyamine oxidation produces H2O2 [hydrogen peroxide] that, in the presence of transition metals, generates a highly toxic ROS [reactive oxygen species], the hydroxyl radical ... in normal non-cancerous cells, up-regulation of polyamine oxidation and H2O2 generation can have undesirable effects”9 such as DNA damage. Polyamines are also growth factors; hence decreasing intracellular polyamines below the optimal intracellular levels for growth is one way to reduce tumor growth and development.9
Trehalose is a natural disaccharide found in many non-mammalian species that protects cells against various environmental stresses (for example, by acting as an osmolyte). Trehalose can be found in our FoldRight™ formulation of osmolytes that act as chaperones to help proteins fold properly. A recent paper1 (thanks again, Will) identifies trehalose as an enhancer of autophagy, a cell-digesting process that, for instance, allows for the clearance of aggregate-prone proteins such as mutant huntingtin fragments (Huntington’s disease), other polyQ mutations, mutant alpha-synucleins (as in Parkington’s and Alzheimer’s diseases), and tau (as in Alzheimer’s). Though trehalose enhances autophagy, it does not do so by inhibiting mTOR.
The recent paper1 notes that, among other things, trehalose inhibits amyloid formation of insulin in vitro and prevents aggregation of beta-amyloid associated with Alzheimer’s disease. The authors explain that the clearance of aggregate-prone proteins such as those mentioned above depends strongly on macroautophagy, generally referred to as autophagy. In this paper, the authors identify a novel role for trehalose as an autophagy inducer, leading to enhanced clearance of aggregation-prone proteins and protecting cells from subsequent pro-apoptotic insults resulting therefrom.
In a new paper,1 researchers report that after acute injury, renal proximal tubular cells (RPTC) recover mitochondrial function by increasing the expression of the master regulator of mitochondrial biogenesis, PGC-1alpha (peroxisome-proliferator-activated-receptor-gamma-coactivator-1alpha). In this study, the authors examined the effects of 5-HT (serotonin) activators (agonists) on mitochondrial biogenesis in RPTC kidney cells, hypothesizing that 5-HT agonists may produce mitochondrial biogenesis in a variety of organs, including kidneys.
DOI, a selective agonist of 5-HT2 was found to increase the activity of PGC-1alpha in RPTC, which express the 2A, 2B, and 2C subtypes of the 5-HT receptor. They report that DOI-induced increases in mitochondrial biogenesis accelerated the recovery in RPTC of mitochondrial function after oxidative injury. They also report that another 5-HT2 receptor agonist m-chlorophenylpiperazine also increased mitochondrial biogenesis. They note that, “[a]lthough DOI is selective for 5-HT2 receptors, EC50 value for DOI [value that activates 50% of receptors] in isolated receptor systems is in the nanomolar range. This observation provides evidence that DOI may act as a more promiscuous activator of 5-HT receptor subtypes in this system and that the observed increases in mitochondrial biogenesis may be through other 5-HT receptors.”
The authors conclude that “... we suggest that DOI or other 5-HT receptor agonists may be an effective therapeutic option to accelerate the recovery of renal function subsequent to acute kidney injury.” Tryptophan (or 5-hydroxytryptophan) is an example of a nutrient that enhances activity of 5-HT (serotonin) receptors, in this case acting as the precursor to serotonin synthesis. The researchers also suggest that 5-HT reuptake inhibitors (SSRIs such as fluoxetine) may act through mitochondrial biogenesis.
Answer: You’d be surprised. A new paper1 reports that a common and readily available prescription drug for athlete’s foot, itraconazole, inhibits both mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) in endothelial cells, suppressing growth and proliferation and, hence, angiogenesis. The closely related drug micronazole is available without a prescription as Micatin™ or Monistat.™ However, we don’t know whether micronazole would work as an angiogenesis inhibitor.
In the introduction to their paper, the authors explain that “[a]mong the upstream signals that are known to affect the mTOR pathway are growth factors, nutrients such as amino acids, cellular energy status, and a variety of environmental stresses.” They note that cholesterol is an important cellular building block. The researchers had screened a library of known drugs in an earlier study looking for inhibitors of angiogenesis and found that one of the most potent hits was an antifungal drug itraconazole. In their followup studies on itraconazole, they have found a link between intracellular cholesterol trafficking and the mTOR pathway in endothelial cells. “Here we report that itraconazole causes blockade of cholesterol egress from endosomal/lysosomal compartments to the plasma membrane, which, in turn, leads to inhibition of both mTORC1 and mTORC2. We provide multiple lines of evidence that mTOR activity in endothelial cells requires proper cholesterol trafficking, adding plasma membrane cholesterol to the list of signal inputs to regulate the mTOR pathway.”
As they explain, “... in the absence of proper cholesterol distribution to the plasma membrane (and likely other intracellular membranes), the mTOR pathway is turned off, leading to the inhibition of endothelial cell [HUVECs, human umbilical vein endothelial cells] proliferation.” This inhibition suggests that the drug may be useful in inhibiting angiogenesis, which requires endothelial cell proliferation. The researchers state that the mTOR inhibitor rapamycin and its analogue temsirolimus both inhibit tumor angiogenesis in vivo and directly inhibit endothelial cell proliferation in vitro. Both mTORC1 and mTORC2 are required for endothelial cell proliferation.1 Skin cancer might be a potential target for therapy with itraconazole, but we don’t know whether effects might differ between the various types of skin cancer, such as basal cell, squamous cell, and melanoma.
The authors also examined the sensitivity of other cell types to the inhibition of the mTOR pathway induced by itraconazole in endothelial cells and found that endothelial cells are, indeed, particularly sensitive to this effect. HeLa cells, for example, were found to be significantly less sensitive to itraconazole than HUVECS.
Note that itraconazole has many drug interactions when taken orally.
That rifle on the wall of the labourer’s cottage or working class flat is the symbol of democracy. It is our job to see that it stays there.— George Orwell
(D&S: Sorry, George. The UK blew it.)
. . . don’t plan your research to fit the supposed dictates of funding agencies—I’ve never done the experiments proposed in any of my grant applications.— Solomon H. Snyder
(D&S: Hmmm. This practice worked out well for outstanding scientists like Dr. Snyder, but it would appear to make the funding somewhat lacking in accountability if everybody can lie about what they plan to do with the money.)
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